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Table of Contents
- Name and Address of Establishment and Employer Identification Number
- Type of Establishment
- Employer's Name and Address
- Establishment Number
- Lines 1 Through 8
- Credit Card Sales
- Rounding Off to Whole Dollars
- Line 1. Total Charged Tips for Calendar Year 2015
- Line 2. Total Charge Receipts Showing Charged Tips
- Line 3. Total Amount of Service Charges of Less Than 10% Paid as Wages to Employees
- Line 4a. Total Tips Reported by Indirectly Tipped Employees
- Line 4b. Total Tips Reported by Directly Tipped Employees
- Line 4c. Total Tips Reported
- Line 5. Gross Receipts From Food and Beverages
- Line 6
- Line 7. Allocation of Tips
- Line 7a. Hours-Worked Method
- Line 7b. Gross Receipts Method
- Line 7c. Good-Faith Agreement
- Line 8. Total Number of Directly Tipped Employees
- Signature
- Who Must Sign
- Example for Line 7b. Gross Receipts Method
- Employer's Optional Worksheet for Tipped Employees
File a separate Form 8027 for each large food or beverage establishment. Use Form 8027-T when filing more than one Form 8027.
Type or print the name and address of the establishment. They may be different from your mailing address, as in the case of employers who have more than one establishment. If mail is not delivered to the street address of the establishment, enter the P.O. box number. The employer identification number (EIN) should be the same as the number on the Forms W-2 that you furnish to the employees and the Form 941 that you file to report wages and taxes for employees working for the establishment.
Check the box (check only one box) on the form that best describes the type of food or beverage establishment.
An establishment that serves evening meals only (with or without alcoholic beverages).
An establishment that serves evening and other meals (with or without alcoholic beverages).
An establishment that serves only meals other than evening meals (with or without alcoholic beverages).
An establishment that serves food, if at all, only as an incidental part of the business of serving alcoholic beverages.
Enter the name and address of the entity or individual whose EIN was provided earlier. Enter foreign addresses as follows: city, province or state, and country. Don't abbreviate the name of the country.
Enter a five-digit number to identify the individual establishments that you are reporting under the same EIN. Give each establishment a separate number. For example, each establishment could be numbered consecutively, starting with 00001. Once you assign a number to an establishment, always use the same number for that establishment each year. If you close an establishment, don't use the number you assigned to it for another establishment.
If the receipts from charges under any credit arrangement (for example, credit or debit cards and other electronic settlement methods) reflect tips, then you must enter on lines 1 and 2 the appropriate amounts shown on the charge receipts. See instructions for line 1 below.
You may round off cents to whole dollars on your Form 8027. If you do round to whole dollars, you must round all amounts. To round, drop amounts under 50 cents and increase amounts from 50 to 99 cents to the next dollar. For example, $1.39 becomes $1 and $2.50 becomes $3.
If you have to add two or more amounts to figure the amount to enter on a line, include cents when adding the amounts and round off only the total.
Enter the total amount of tips that are shown on charge receipts for the year.
Enter the total sales (other than nonallocable receipts as defined earlier) from charge receipts that had a charged tip shown. Include credit card charges and other credit arrangements and charges to a hotel room unless your normal accounting practice consistently excludes charges to a hotel room. Don't include any state or local taxes in the amounts reported.
Enter the total amount of service charges of less than 10% that have been added to customers' bills and have been distributed to your employees for the year.
Enter the total amount of tips reported for the year by indirectly tipped employees, such as cooks, bussers, and service bartenders. Indirectly tipped employees generally receive their tips from other tipped employees and not directly from the customer.
Enter the total amount of tips reported for the year by directly tipped employees, such as bartenders and waitstaff. Directly tipped employees receive tips directly from customers.
Add the amounts on lines 4a and 4b and enter the result on line 4c. This amount can't be a negative amount.
|Line 4a
|Line 4b
|Line 4c
Enter the total gross receipts from the provision of food and beverages for this establishment for the year.
If you don't charge separately for providing food and beverages along with other goods or services (such as a package deal for food and lodging), make a good-faith estimate of the gross receipts from the food and beverages. This estimate must reflect the cost to the employer for providing the food and beverages plus a reasonable profit factor.
Enter the result of multiplying line 5 by 8% (.08) or a lower rate (if the establishment was granted a lower rate by the IRS).
If a lower rate was granted, write the rate in the space provided and attach a copy of the IRS determination letter. If you file Form 8027 electronically, see Pub. 1239 for instructions on submitting a copy of the IRS determination letter.
If the amount shown on line 6 is more than the amount of tips reported by your employees on line 4c, you must allocate the excess to those employees. Enter the excess on line 7. There are three methods by which you may allocate tips. Check the box on line 7a, 7b, or 7c to show the method used.
Establishments that employ fewer than the equivalent of 25 full-time employees (both tipped and nontipped employees) during a payroll period may use the hours-worked method to allocate tips. You will be considered to have employed fewer than the equivalent of 25 full-time employees during a payroll period if the average number of employee hours worked (both tipped and nontipped employees) per business day during a payroll period is less than 200 hours.
To allocate tips by the hours-worked method, follow the steps explained in Line 7b. Gross Receipts Method below. However, for the fraction in step 3 of the gross receipts method, substitute in the numerator (top number) the number of hours worked by each employee who is tipped directly, and in the denominator (bottom number) the total number of hours worked by all employees who are directly tipped for the payroll period. See Regulations section 31.6053-3(f)(1)(iv) for details.
If you use the hours-worked method, be sure to enter on line 7a the average number of employee (both tipped and nontipped) hours worked per business day during the payroll period. If the establishment has more than one payroll period, you must use the payroll period in which the greatest number of workers (both tipped and nontipped) were employed.
If no good-faith agreement (as explained later) applies to the payroll period, you must allocate the difference between total tips reported and 8% of gross receipts using the gross receipts method (or hours-worked method (line 7a)) as follows (see Example for Line 7b. Gross Receipts Method, later).
Multiply the establishment's gross receipts (other than nonallocable receipts) for the payroll period by 8% (.08) or the approved lower rate.
Subtract from the amount figured in step 1 the total amount of tips reported by employees who were tipped indirectly for the payroll period. This difference is the directly tipped employees' total share of 8% (or the lower rate) of the gross receipts of the establishment. Indirectly tipped employees don't receive tips directly from customers. Examples are bussers, service bartenders, and cooks. Directly tipped employees, such as waitstaff and bartenders, receive tips directly from customers. Employees, such as maitre d's, who receive tips directly from customers and indirectly through tip splitting or pooling, are treated as directly tipped employees.
For each employee who is tipped directly, multiply the result in step 2 by the following fraction: the numerator (top number) is the amount of the establishment's gross receipts attributable to the employee, and the denominator (bottom number) is the gross receipts attributable to all directly tipped employees. The result is each directly tipped employee's share of 8% (or the lower rate) of the gross receipts for the payroll period.
From each directly tipped employee's share of 8% or the lower rate of the gross receipts figured in step 3, subtract the tips the employee reported for the payroll period. The result is each directly tipped employee's shortfall (if any) for the period.
From the amount figured in step 1, subtract the total tips reported by both directly and indirectly tipped employees. The result is the amount that has to be allocated among the directly tipped employees who had a shortfall for the payroll period as figured in step 4.
For each directly tipped employee who had a shortfall for the period as figured in step 4, multiply the amount in step 5 by the following fraction: the numerator is the employee's shortfall (figured in step 4), and the denominator is the total shortfall of all directly tipped employees. The result is the amount of allocated tips for each directly tipped employee.
An allocation can be made under a good-faith agreement. This is a written agreement between you and at least two-thirds of the employees of each occupational category of employees who receive tips (for example, waitstaff, bussers, and maitre d's) working in the establishment when the agreement is adopted. The agreement must:
Provide for an allocation of the difference between total tips reported and 8% (or the lower rate) of gross receipts among employees who receive tips that approximates the actual distribution of tip income among the employees;
Be effective the first day of a payroll period that begins after the date the agreement is adopted, but no later than January 1 of the next year;
Be adopted when there are employees in each occupational category who would be affected by the agreement; and
Allow for revocation by a written agreement adopted by at least two-thirds of the employees in occupational categories affected by the agreement when it is revoked. The revocation is effective only at the beginning of a payroll period.
Keep a copy of the good-faith agreement for your records.
Enter the total number of directly tipped employees who worked at the establishment during 2015. This is the cumulative total of all directly tipped employees who worked at the establishment at any time during the year. If you have a large turnover of directly tipped employees, this number may be large. Don't use this number to determine if you must file Form 8027. Instead, see the Worksheet for Determining if You Must File Form 8027 for Calendar Year 2015, earlier.
Sign your name and include your title. Then enter the date signed and the best daytime telephone number where the IRS can reach you, including area code.
The following persons are authorized to sign the return for each type of business entity.
A large food or beverage establishment has gross receipts for a payroll period of $100,000 and has tips reported for the payroll period of $6,200. Directly tipped employees reported $5,700, while indirectly tipped employees reported $500.
|Directly tipped employees
|Gross receipts for payroll period
|Tips reported
|$100,000 (gross receipts) x .08 = $8,000
|$8,000 - $500 (tips reported by indirectly tipped employees) = $7,500
|Directly tipped employees
|Directly tipped employees' share of 8% of the gross
Gross receipts ratio
|Employee's share of 8% of gross
|18,000/100,000 =
|16,000/100,000 =
|23,000/100,000 =
|17,000/100,000 =
|12,000/100,000 =
|14,000/100,000 =
|Directly tipped employees
|Employee's share of 8% of the gross
Tips reported
|Employee shortfall
|$1,080 =
|880 =
|1,810 =
|800 =
|$ 900
|450 =
|680 =
|Total shortfall
|$8,000 less $6,200 (total tips reported) = $1,800 (amount allocable among employees who had a shortfall)
|Shortfall employees
|Allocable amount
Shortfall ratio
|Amount of allocation
|$270/1,885 =
|320/1,885 =
|475/1,885 =
|450/1,885 =
|370/1,885 =
|Since employee C has no shortfall, there is no allocation to C.
Unreported tip income can lead to additional employer liability for social security tax and Medicare tax. As a means of determining if your employees are reporting all of their tips to you, please take a few minutes to voluntarily complete the following worksheet. Completing this worksheet is only for the employer's information (Don't send it to the IRS).
|Enter amount from Form 8027, line 1
|Enter amount from Form 8027, line 2
|Divide line 1 by line 2, enter as a decimal (at least 4 decimal places)
|Enter amount from Form 8027, line 4c
|Enter amount from Form 8027, line 5
|Divide line 4 by line 5, enter as a decimal (at least 4 decimal places)
|Subtract line 6 from line 3; if zero or less, stop here
|Potential unreported tips. Multiply line 7 by line 5
If the entry on line 7 is zero or less, your employees are probably accurately reporting their tips; however,
If the entry on line 8 is greater than zero, depending on the type of operation you have and whether or not you have allocated tips, it is possible that your employees aren't reporting all of their tip income to you.
If it appears that not all tips are being reported to you, the IRS offers a service called the Tip Rate Determination & Education Program. This program can assist you, the employer, in implementing more effective methods of tip income reporting. The program also offers assistance in educating tipped employees concerning their obligations relating to the reporting of any tip income they receive. To find out more about this program or to participate in a voluntary tip compliance agreement, visit IRS.gov and type "restaurant" in the search box. You may also send an email to email@example.com and request information on this program.
An information reporting customer service section is available to answer questions about reporting on Forms 8027, W-2, W-3, 1099, and other information returns. If you have questions about reporting on information returns, call:
1-866-455-7438 (toll free),
(304) 263-8700 (toll call), or
(304) 579-4827 (TDD/TTY for persons who are deaf, hard of hearing, or have a speech disability).
You can also reach the center by email at email@example.com. Don't include tax identification numbers (TINs) or attachments in email correspondence because it isn't secure.
|More Online Instructions


text: http://www.talkandroid.com – We first spotted Acer's affordable 7-inch Iconia B1 tablet last month, where it was rumored the device would launch globally at a staggering $99 price point. Well, new information has leaked out today suggesting the device would in fact launch as the Iconia B this month. The tablet is slated to make its world debut in India for 7,999 INR, which equates to around $150 US, a bit more than originally rumored. If you're unfamiliar, the Acer Iconia B runs Android 4.1.2 Jelly Bean and sports a 7-inch 1024 x 600 display, a 1.2-GHz dual-core MediaTek processor and 512MB of RAM. U (IT news)

subset: commoncrawl


text: Bilal Powell #29 and Sheldon Richardson #91 of the New York Jets celebrate a touchdown (Mike Ehrmann/Getty Images) [ Jets' Finale Eliminates Dolphins, 20-7 By Associated Press December 29, 2013 The Miami Dolphins were eliminated from the scramble for the AFC's final wild-card berth Sunday by the Jets, who thrived in the role of spoilers against their archrivals and won 20-7. Read More|Comment Category: News, Sports Tags: 12/29 Games, Jets, New York Jets Send to a friend! Share on Facebook Share on Twitter Pin it! Reddit This! Share on Tumblr!


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NPR1 is a gene of Arabidopsis thaliana required for the perception of salicylic acid. This perception triggers a defense response and negatively regulates the perception of jasmonates. Surprisingly, the application of methyl jasmonate also induces resistance, and NPR1 is also suspected to be relevant. Since an allelic series of npr1 was recently described, the behavior of these alleles was tested in response to methyl jasmonate.
The response to methyl jasmonate of different npr1s alleles and NPR1 paralogs null mutants was measured by the growth of a pathogen. We have also tested the subcellular localization of some npr1s, along with the protein-protein interactions that can be measured in yeast. The localization of the protein in npr1 alleles does not affect the response to methyl jasmonate. In fact, NPR1 is not required. The genes that are required in a redundant fashion are the BOPs. The BOPs are paralogs of NPR1, and they physically interact with the TGA family of transcription factors.
Some npr1 alleles have a phenotype in this response likely because they are affecting the interaction between BOPs and TGAs, and these two families of proteins are responsible for the resistance induced by methyl jasmonate in wild type plants.
Keywords:Methyl jasmonate; Salicylic acid; Arabidopsis; NPR1; BOPs; Defense
Plants are constantly defending themselves against pathogens by means of a wide array of mechanisms. Some of them are pre-existing (or non inducible) and others are induced in response to the pathogen attack. Salicylic acid (SA, reviewed by [1]) is a plant hormone which is crucial for the inducible response of Arabidopsis thaliana (Arabidopsis) to biotrophic pathogens like Pseudomonas spp[2]. When a pathogen is perceived, SA is produced and accumulated, producing a proper defense. This SA signaling occurs not only where the attack takes place, since defense is also enhanced in leaves different from the one inoculated. This is called Systemic Acquired Resistance (SAR, [3]). SA has an intricate crosstalk with other hormones, showing an overall negative crosstalk with auxins, ethylene (ET), and jasmonates (JA, crosstalk of hormones reviewed by [4]). In the case of JA, it has been shown that the active form in planta is JA-Ile (reviewed by [5]), while in the laboratory is used exogenously as Methyl Jasmonate (MeJA).
NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) is the main gene required for SA perception [6]. There are five paralogs of NPR1 in Arabidopsis [7], BLADE-ON-PETIOLE1 (BOP1) and BOP2 have an important role in development [8], NPR3 and NPR4 have a role in defense [9], probably through SA perception [10], and no specific function for NPR2 has been described, besides a secondary role in SA perception [11]. There are other genes that are relevant for signal transduction, like the family of TGA transcription factors whose products interact with NPR1 [12], but they are required in a redundant fashion.
NPR1 has been described as having more than one role in defense, since it is also important in the Induced Systemic Resistance (ISR, [13]). ISR is defined as the resistance triggered at the leaves by a non pathogenic organism inoculated in the roots, and while SAR requires SA signaling, ISR requires MeJA and ET signaling. As with SA, exogenous applications of MeJA and ET trigger resistance in Arabidopsis towards some biotrophs, like Pseudomonas[14]. It has been proposed that NPR1 is relevant for the resistance-inducing ability of MeJA ([13], hereafter abbreviated as RIM), although RIM it is not necessarily equivalent to ISR. While the role of NPR1 in SA perception takes place in the nucleus [15], its function in RIM is not so clearly understood. It has been described a cytosolic function of NPR1 crucial in cross-talk between SA and JA signaling [16]. Furthermore, Arabidopsis transcriptome analysis upon pathogen infection has suggested that such cytosolic function is also involved in the modulation of JA-dependent defenses [17]. The npr1-3 mutant, which produces a truncated cytoplasmatically localized npr1 protein with no nuclear localization signal, has been reported to be affected only in SA-dependent gene expression, not in JA and ET dependent genes. In contrast, the npr1-1 mutant, which has a mutation in a key domain, is affected in the expression of SA, JA and ET-dependent genes [17]. More recent studies support such cytosolic NPR1 function as regulator of JA-dependent defense responses ([18-20]).
BOP1 was first identified by its mutant phenotype of ectopic blades along the petioles, as well as some alterations in the flowers [8]. The first allele identified was a dominant negative, since T-DNA insertions in bop1 did not reproduce the phenotypes of ectopic blades [21]. Once BOP1 was identified as paralog to NPR1[22], it was shown that other paralog, BOP2, functions redundantly with BOP1[21]. The double mutant bop1 bop2 reproduced all the developmental phenotypes of ectopic blades, but it was wild type when inoculated with Pseudomonas[21], and it is also wild type for SA perception [23].
Since a collection of npr1 alleles has recently been available [11], we tested the hypothesis that the role of NPR1 in RIM is cytosolic. In this work, we show that NPR1 has no role in RIM in wild type conditions, since the genes responsible for RIM are BOP1 and BOP2, with an important part being played by the TGAs. Therefore, two genes required for the normal development of the leaf, are also required for plant defense.
Role of NPR1 in RIM
NPR1 has been characterized as a result of observing the response to SA of the great number of alleles described for it [24]. NPR1 has also been described as essential for RIM, but there are differences between alleles, since npr1-1 and npr1-3 have different RIM ([20,25]). npr1-1 and npr1-3 have other differences in phenotypes related to MeJA. Thus, the SA-JA antagonism is not present in npr1-1, but it is active in npr1-3 [16]. Other difference is the gene expression, whereas npr1-1 was affected in SA, JA, and ET dependent genes upon Pto inoculation, npr1-3 was only affected in SA dependent genes [17]. These different phenotypes have been attributed to the lack of nuclear localization in npr1-3, since the truncated cytosolic protein would be functional to modulated JA-dependent defense response [19].
In order to determine the precise role of NPR1 in RIM, and to asses the role of the cytosolic vs. nuclear localization, we tested an allelic series of 43 npr1 alleles, defined by their inability to perceive SA [11]. The npr1 alleles produced a mixture of phenotypes in RIM (Figure 1), and out of the 43 alleles tested, 11 had a significant RIM (RIM+, Figure 1a), meaning that the growth of Pto in MeJA treated plants was significantly lower than in mock treated plants (Student's test of one tail, with P<0.05). The rest of alleles showed no response (RIM-, 11 out of 32 are shown in Figure 1b). These two categories of alleles did not share any obvious feature and had the same proportion of stops and point mutations in each category. In fact, in the RIM+ alleles tested, six point mutations were widely scattered along the protein (Figure 1c, npr1-3 is included as reference).
Figure 1. npr1 alleles differ in their response to MeJA. Forty-three npr1 alleles were treated with 100 μM methyl jasmonate (MeJA), with 0.1% DMSO and 0.02% Silwet L-77 or a mock treatment. One day later, Pseudomonas syringae pv. tomato DC3000 (Pto) was inoculated and its growth measured three days later in a logarithmic scale. For each genotype and treatment three samples with 5 plants per sample were taken. The bars show the average ± SD and npr1-1 and npr1-3 are included as controls for negative and positive response to MeJA respectively. (a) npr1 alleles that show resistance induced by MeJA. (b) npr1 alleles that do not show resistance induced by MeJA. (c) Schematic representation of the npr1 alleles that showed resistance induced by MeJA along the structure of NPR1. BTB/POZ stands for
- Po
These alleles could somehow affect the localization of the protein inside the cell, even if the mutation was not in the NLS. To check this possibility, cDNAs of three RIM+ alleles (npr1-22, -35 and −44, Additional file 1), and three RIM- alleles (npr1-1, -40, and −56, Additional file 1), chosen among the point mutations, were cloned along with the wild type NPR1. Then, these seven cDNAs were transiently expressed in N. benthamiana under the control of the 35S promoter and with the GFP marker. The free GFP was detectable in the nucleus and in the cytoplasm. But when the wild type and the six alleles of npr1 were expressed in the same conditions, GFP was detected mainly in the nucleus, with no difference existing between the two classes of alleles (Figure 2a).
Additional file 1. Figure S1. Localization of cloned npr1 alleles mentioned in the text. Figure S2. NPR1HBD treated with DEX is more sensitive to BTH. Figure S3. Expression levels of NPR1 paralogs. Figure S4. Response of bop1 bop2 to SA and BTH. Table S1. List of primers used.
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Figure 2. The nuclear localization of npr1 alleles is not relevant for resistance induced by MeJA. (a) Transient expression of GFP, NPR1:GFP, and six npr1 alleles (three from Figure 1a and three from Figure 1b). Agrobacterium tumefaciens containing the mentioned genes under the promoter 35S were infiltrated in leaves of Nicotiana benthamiana, and the expression was detected with a confocal microscopy four days later. (b) The cytoplasmic anchoring of NPR1 does not complement npr1-1 in its response to MeJA. Plants with the transgene 35SCaMVp:NPR1:HBD (abbreviated as 3NH) in npr1-1 or npr1-3 background and its controls were treated with either dexamethasone (DEX) or mock solution, and then treated with either MeJA or mock solution. One day later, Pto was inoculated and measured as described in Figure 1. One asterisk means a significant difference with P<0.05, and two asterisks means P<0.01 (Student's test of one tail).
As a complementary approach, we took advantage of the transgenic line that overexpresses NPR1 fused to the steroid hormone binding domain of the rat glucocorticoid receptor (HBD, and the transgenic plants are known as NPR1-HBD, [15]). NPR1-HBD remains exclusively in the cytosol in mock conditions and should be functional in RIM. The original line is in an npr1-3 background (RIM+), and therefore the transgene was transferred to an npr1-1 background (RIM-) to check for complementation. Treatments with BTH and with and without glucocorticoid dexamethasone (DEX) showed that NPR1-HBD was functional (Additional file 1). NPR1-HBD, even under the control of the 35S promoter, did not complement the lack of RIM in npr1-1 (Figure 2b). When DEX was applied, NPR1-HBD moved to the nucleus and npr1-1 was complemented in the RIM phenotype. Note that the presence of cytosolic NPR1-HBD in an npr1-3 background did not enhance RIM in comparison to npr1-3 alone.
The npr1 alleles RIM+ or RIM- did not share any obvious feature, so it would be difficult to assign a precise role to the wild type gene. A critical genetic resource to discern the role of a gene is the null allele. Therefore, the response of two null alleles of npr1 to RIM was measured (Figure 3a). Both npr1-70 and npr1-71 are in Laer-0 background, so an introgression of npr1-1 in Laer-0 was used as control [11]. These two null alleles responded to RIM in all experiments like the wild type. npr1-70 introgressed in Col-0 responded again like the wild type, which ruled out any ecotype effect (Figure 3b). Since the direct role of NPR1 in RIM was in question as a consequence of the aforementioned results, we included an independent RIM- control, coi1-40 (see Methods).
Figure 3. NPR1 is not necessary for resistance triggered by MeJA. (a) Two null npr1 alleles (npr1-70 and npr1-71, both in Laer-0 background), plus their controls were tested for resistance induced by MeJA as described in Figure 1. coi1-40 is introduced as negative control for resistance triggered by MeJA. npr1-1L is npr1-1 introgressed in Laer-0 [11]. (b) Introgressed lines of npr1-70 in Col-0 show the same phenotype than the original npr1-70. (c) The effect of some npr1 alleles on resistance triggered by MeJA is not due to an excess of salicylic acid. Double mutants of npr1-1 with NahG, eds5, and sid2 were tested for their response to MeJA in resistance. One asterisk means a significant difference with P<0.05, and two asterisks means P<0.01 (Student's test of one tail).
The role of NPR1 in this response might be indirect. Thus, one scenario would be a reinforcement of the negative crosstalking between SA and MeJA. npr1 alleles produced more SA when infected with Pto[26] and seemed unable to metabolize it [27]. RIM- alleles -defective in terms of SA perception- might have left intact the negative crosstalk between SA and MeJA, and an excess of SA repressed the response to MeJA beyond the wild type levels. Therefore, the RIM+ alleles would be defective in terms of both SA perception and SA-MeJA crosstalking, an explanation that would also be in agreement with the behavior of the null alleles.
To test this hypothesis, the double mutants between npr1-1 and NahG (a transgenic plant that degrades SA, [28]), eds5 (a mutant in SA transport, [29]), and sid2 (a mutant in SA biosynthesis, [30]), were constructed and tested for RIM. npr1-1 did not respond to MeJA even if the levels of SA were low (Figure 3c), so the hypothesis of a reinforcement of the negative crosstalk was not supported.
BOP1 and BOP2 and their role in RIM
The experiments with the null alleles showed that NPR1 was not necessary for RIM. Perhaps NPR1 and other gene(s) would be redundantly responsible of RIM, and while null npr1 alleles would have a RIM+ phenotype, some npr1 alleles could be RIM- by interacting with other protein(s) negatively. The most likely candidates for these interactions were the NPR1 paralogs, since their proteins shared the same overall structure. There are five additional paralogs of NPR1 in the Arabidopsis genome [7], and we analyzed double mutants of npr1-70 with different paralogs (Figure 4). There was no proof of NPR1 having a redundant role in RIM. NPR2 did not play a significant role in RIM (Figure 4a) and the same was true for the double npr3 npr4 (Figure 4b,c and [7]). Strikingly, the double bop1 bop2[21] was RIM- (Figure 4d).
Figure 4. A role for the NPR1 paralogs in resistance triggered by MeJA. Double mutants of npr1-70 with NPR1 paralogs and their corresponding controls were tested for resistance triggered by MeJA as described in Figure 1. (a) Double mutants with npr2. (b)npr3. (c)npr4. (d)bop1 and bop2. Four independent lines were tested in each case, except with bop1, where only two were obtained. One asterisk means a significant difference with P<0.05, and two asterisks means P<0.01 (Student's test of one tail).
Single bop1 and bop2 were also tested for RIM and showed to be wild type (Figure 5a). BOP1 and BOP2 exert their function in part through transcriptional regulation of KNAT6 and physical interaction with PAN (TGA8, [31]), but T-DNA insertions predicted to disrupt KNAT6 or PAN activity did not have an effect on RIM (Figure 5a). To rule out the possibility that other mutations besides bop1 and bop2 were producing this RIM- phenotype, we constructed an artificial microRNA (amiRNA, [32]) to deplete the levels of BOP1 and BOP2 at the same time. Eight independent homozygous transgenic lines for amiRNA (BOP1 - BOP2) were analyzed for RIM (Figure 5b). Five out of eight lines were RIM-, and the remaining three responded less than the wild type control. The levels of both genes were partially depleted in the eight lines (Figure 5c); five of the lines had both genes significantly reduced, and all had BOP2 significantly reduced. None of these lines had the characteristic blade-on-petiole macroscopic phenotype, not even as the subtle phenotype of bop1 alone.
Figure 5. Only the reduction of both bop1 and bop2 expression affects resistance triggered by MeJA. (a) The double bop1 bop2, the single mutants, T-DNAs insertions in KNAT6 and PAN1, and their controls were tested for resistance induced by MeJA as described in Figure 1. KNAT6 and PAN1 are genes that interact with BOP1 and BOP2. (b) Reduction of BOP1 and BOP2 expression partially phenocopies the double bop1 bop2. Eight independent transgenic lines of an artificial micro RNA designed to reduce the levels of BOP1 and BOP2 (amiRNA(BOP1-BOP2) were tested as described in Figure 1. The lines did not show any macroscopic blade-on-petiole phenotype. (c) RNA was extracted from 3-week-old plants of the lines described in (b), and transcript levels for BOP1 and BOP2 were measured by means of RT-qPCR. Levels of expression are normalized to three reference genes and to the level of Col-0 in mock. Asterisks mark the significance of the difference between the levels of expression of each line with Col-0; one asterisk means a significant difference with P<0.05, and two asterisks means P<0.01 (Student's test of one tail).
The previous experiments had shown that BOP1 and BOP2 were acting redundantly in RIM (Figure 4d). Therefore, increasing the amount of any of them should have an effect on RIM, especially since normal levels of BOP1 and BOP2 are quite low (Additional file 1, [21]). The overexpression lines of BOP1 and BOP2 described [33] were analyzed for RIM. 35S:BOP1 had a stronger RIM than Col-0, and 35S:BOP2 had a strong variation in the MeJA treated plants (Figure 6a). At the time of the experiments, each population looked homogeneous, but when these plants were grown to set seeds, two phenotypes could be observed in each transgenic line. Approximately half of the plants showed a wild type phenotype, and the other half reproduced the dwarf plants described [33]. Seeds from both lines and from both phenotypes reproduced the two phenotypes. It seems that it was an issue of silencing, since RNA taken from plants classified by their mentioned phenotype diverged widely in the transgene expression (Figure 6b).
Figure 6. The overexpression of BOP1 or BOP2 produces more response to MeJA. (a) 35S:BOP1 and 35S:BOP2 lines [33] and their controls were tested as described in Figure 1. One asterisk means a significant difference with P<0.05, and two asterisks means P<0.01 (Student's test of one tail). (b) Silencing of the overexpression lines. 35S:BOP1 and 35S:BOP2 lines were phenotyped at the time of bolting as having a characteristic phenotype (Phen.) [33] or being wild type (No Phen.). Then, RNA was extracted from several plants and the levels of the transgenes quantified as described in Figure 5c. On the left side of the plot, relative expression of BOP1 and, on the right, relative expression of BOP2. Means with the same letter are not significantly different (Fisher's LSD test, P<0.05). The test was performed separately for each gene.
bop1 bop2 specificity in RIM
The bop1 bop2 double mutant lacked a RIM response, but this failure in MeJA signaling might occur at different points of the signal transduction. For example, the defect could target a general signaling component affecting all MeJA responses (e.g. coi1,[34]), or a specialized part of the pathway, affecting a subset of MeJA responses (e.g. jin1,[35]). When bop1 bop2 plants were grown in plates containing MeJA, the growth of the roots was similar to the wild type controls (Figure 7a). Other phenotypes of bop1 bop2 plants growing in MeJA plates were similar to the wild type controls (carotenoids production, size of aerial part, number of trichomes, etc.; data not shown). Another effect of MeJA is the increase of senescence in detached leaves, measured as chlorophyll production [36]. bop1 bop2 responded as the wild type in this particular system (Figure 7b). Coronatine is a virulence factor of several Pseudomonas isolates with structural and functional similarities to JA-Ile (the functional form of Jasmonate in planta, [5]), therefore a mutant Pto that lacks coronatine [37] grows less in Col-0 than the wild type Pto. bop1 bop2 was also wild type in response to Pto with and without coronatine (Figure 7c). Inoculations with Plectospharaella cucumerina, a fungus that causes more disease in MeJA mutants than in wild type plants [38], did not cause any more disease in bop1 bop2 than in Col-0 (data not shown). If bop1 bop2 was not a MeJA signaling mutant, but specifically in RIM, it could be defective in the other signaling required for ISR; ET. It was not; when 1 mM of 1-aminocyclopropane-1-carboxylic acid (ACC, an ET precursor) was sprayed to bop1 bop2, the resistance triggered was similar to that triggered in the wild type controls (Figure 7d). etr1 (ETHYLENE RESPONSE 1,[39]) was included as a negative control of resistance induced by ethylene.
Figure 7. The double bop1 bop2 is specifically affected in the resistance induced by MeJA. (a) Length of primary root. bop1 bop2 and their controls were grown in plates with Johnson's Media [58] with or without 50 μM MeJA. At the age of 10 days, the lengths of the roots were measured in both conditions and their ratio (MeJA treated divided by mock treated) expressed as percentage. Means with the same letter are not significantly different (Fisher's LSD test, P<0.05). (b) Senescence induced by MeJA. The indicated genotypes were grown in soil and mature leaves from 6-week-old plants were cut and floated on water with or without 100 μM MeJA. The amount of chlorophyll (in μg/g fresh weight) was measured after four days of darkness, with three groups of leaves of c. 1 g each. (c) Coronatine as a virulence factor. Bacteria with coronatine (Pto, COR+) or without coronatine (Pto(cfa–), COR-) were inoculated and their growth measured as in Figure 1. (d) Resistance induced by ethylene. The plants were treated with 1 mM 1-aminocyclopropane-1-carboxylic acid (ACC) or a mock treatment, and then Pto was inoculated and measured as in Figure 1. etr1 is a negative control of resistance induced by ethylene. One asterisk means a significant difference with P<0.05, and two asterisks means P<0.01 (Student's test of one tail).
NPR1 and BOPs interactions
Once it was clear that both BOP genes are required for RIM, we tested the model that npr1 RIM- alleles could have a dominant negative effect on BOP activity, either directly or indirectly. To first test whether NPR1 had an effect in the interaction between BOP proteins, we used a yeast two-hybrid assay. As reported, BOP1 and BOP2 interacted with each other [40]. Next, we introduced in a third plasmid containing wt NPR1 or various mutant npr1 alleles presented in Figure 2a. If the effect of npr1 on RIM were a direct interaction between NPR1 and the two BOPs, the alleles that diverge in their RIM phenotype would diverge in their ability to interfere in the interaction of BOP1 and BOP2. The two classes of npr1 alleles did not have a distinct behavior (Figure 8a, the first three npr1 alleles are RIM-, and the last three are RIM+), therefore the dominant negative effect did not seem to be direct.
Figure 8. The TGA family plays an important role in resistance induced by MeJA. (a) Interaction of BOP1 and BOP2 in the yeast two hybrid system in presence of several npr1 alleles cloned in a third plasmid. NPR1 is included as control, the next three alleles do not respond to MeJA in defense, and the remaining three respond as wild type. The interaction is measured in Miller Units [55]. (b) Null alleles of several TGAs alleles were analyzed as in Figure 1. tga2,5,6 stands for the triple tga2 tga5 tga6. One asterisk means a significant difference with P<0.05, and two asterisks means P<0.01 (Student's test of one tail). (c) Interaction between BOP1 and TGA2, and TGA6 in the yeast two hybrid system in the presence of the same npr1 alleles of (a). Means with the same letter are not significantly different (Fisher's LSD test, P<0.05).
All the NPR1 paralogs tested interact with members of the TGA family in a different degree [7,21]. Therefore, the TGAs would be a reasonable candidate for being the third component, and their interaction with RIM- alleles would indirectly affect the function of BOP1 and BOP2. As a control, single mutants in TGA1 and TGA7 produced a significant RIM (Figure 8b), but when three specific tgas are knocked out at the same time (a triple which phenocopies an npr1 mutant in SA response, [41]), there is no RIM (Figure 8b).
We reasoned that one or several of these three TGAs (TGA2, 5, and 6) might have a functional interaction with the BOPs, which might be affected by the RIM- alleles. To test this hypothesis BOP1 and each of the mentioned TGAs were introduced in the yeast two-hybrid system with the npr1 alleles mentioned above in a third plasmid. TGA2 and TGA6 interact differentially with BOP1 depending on the npr1 protein present (Figure 8c). There was an enhancement of the interaction in two out of the three RIM- alleles, and no interference in two out of the three RIM+ alleles. The interaction TGA5-BOP1 was not affected by the presence of npr1 proteins (data not shown). The experiments were repeated with BOP2 producing similar results (data not shown). In sum, the data indicated that BOPs and TGA2, TGA5 and TGA6 are required for RIM, that BOPs interact with these (and other) TGAs, and that NPR1 may modulate the affinity or stability of the interactions.
NPR1 is not required for RIM
NPR1 is an essential gene for SAR and SA perception [6]. npr1-1, the most widely used allele, is also impaired in RIM [13]. We speculated that since npr1-3 is wild type for RIM ([20,25]), and it has been reported that the difference of some phenotypes between npr1-1 and npr1-3 was due to the lack of NLS in npr1-3 ([17,19]), the same could be true for RIM. However, we show here that the nuclear localization of the alleles makes no difference. This conclusion is supported by multiple lines of evidence. First, the npr1 alleles with RIM+ are not structurally similar to npr1-3, since not all of them are affected in the NLS (Figure 1c). Even an allele with a point mutation in the NLS (npr1-22, Additional file 1) should be partially localized in the nucleus [15]. Second, three RIM- and three RIM+ alleles do not differ in their nuclear localization or stability when transiently expressed in N. benthamiana (Figure 2a, Additional file 1). While these proteins are no longer functional, they respond to the signals of a wild type background by localizing in the nucleus. Third, when a functional NPR1 is anchored in the cytoplasm there is no complementation of the RIM- phenotype in an npr1-1 background (Figure 2b), nor there is an increase in RIM phenotype in an npr1-3 background. In fact, the application of DEX triggered an increased RIM in both backgrounds (discussed below).
But, most importantly, NPR1 is not required for RIM, since the null npr1 alleles are RIM+ regardless of the background (Figure 3a,b). We also discarded that NPR1 could be a part of RIM in a redundant fashion with its paralogs (Figure 4).
An interesting alternative for the role of NPR1 in RIM would be an effect on the crosstalking between SA and MeJA. NPR1 has been described as a key point in the negative regulation between SA and MeJA. Thus, the RIM+ alleles could be defective in both SA perception and in SA-MeJA crosstalk, while the RIM- alleles would be defective only in SA perception but not in SA-MeJA crosstalk. The inoculation with Pto triggers an increase in the levels of SA, and in the case of the npr1 alleles, there is more SA than in the wild type [42]. Although this hypothesis would explain the phenotype of the null alleles, it was rejected after the experiment of Figure 3c, where a severe reduction of SA levels in a RIM- allele did not have any effect on the phenotype.
BOP1 and BOP2 are redundant in RIM
The redundant functions of BOP1 and BOP2 are essential for normal development. Previous work has shown that the double mutant has numerous defects in plant architecture including altered leaf morphology [43], changes in floral patterning [21], defects in the conversion of shoots to flowers [44] and loss of floral-organ abscission [45]. The double mutant was tested for basal defense [21] and SA perception (Additional file 1) but no difference from wild type was found. We show here that both genes are also redundantly required in defense against pathogens triggered by MeJA. Interestingly, whereas significant loss of BOP activity is required to exert changes in development [21], RIM is abolished in plants that are only partially silenced for the BOP genes (Figure 5b,c). Thus, the levels of gene expression required for RIM are higher than those required for normal development. Compatible with this idea, BOPs expression in plants is highly localized, restricted to young organ primordia, leaf petioles, and lateral organ boundaries, which may make systemic responses to MeJA sensitive relatively minor changes in BOP transcript abundance. Both NPR1 and the BOPs localize to the cytoplasm as well as nucleus and interact with members of the TGA family of bZIP transcription factors, albeit with different affinities (e.g., [12]). In development, BOP1 and BOP2 form a nuclear complex with TGA8/PERIANTHIA (PAN) to regulate number of sepals and petals in flowers and potentially to promote floral meristem fate [21]. Given that pan loss-of-function did not reproduce the RIM- phenotype (Figure 5a) other genes, perhaps TGAs, are involved in this phenotype, as shown for SA perception [23]. Given that BOPs play both positive and negative roles in transcriptional regulation of the KNOX (Knotted1-like homeobox) gene KNAT6[46], we also tested if RIM was affected by knat6 loss-of-function, but again, no difference was observed (Figure 5a). This may reflect redundancy with other KNOX genes, or more likely, that BOP regulation of RIM is independent of KNAT6.
Whether bop1 bop2 recapitulates or not all the phenotypes of the RIM- npr1 alleles (e.g. ISR, [13]; Verticilium resistance, [18]; resistance induced by Piriformospora indica,[47]; etc.) remains to be assessed. We did check that there were similar phenotypes in the specificity of response to MeJA as well as the fact that bop1 bop2 was wild type for the rest of MeJA phenotypes (Figure 7). But there were strong differences, since npr1-3 is affected in basal defense and SA perception while bop1 bop2 is wild type for both phenotypes [21]. Regarding ET, the other hormone relevant for ISR, it has been proposed that applications of this hormone could render the crosstalk between SA and MeJA independent of NPR1 [19]. It seems plausible that the ET induced resistance works as the MeJA induced resistance and other proteins -perhaps NPR1 paralogs, but not the BOPs (Figure 7d) - might also be affected by some alleles of npr1.
Some npr1 alleles interfere in the BOPs-TGAs interaction
The RIM- npr1 alleles were the majority of the alleles found (32 RIM- vs. 11 RIM+). How is this compatible with the fact that the null npr1 alleles are RIM+? A possible explanation was the selection used in the screening. Since the selection was made for complete loss of SA perception, perhaps most of the RIM+ alleles had a phenotype of partial SA perception, as the null alleles. Then, the prediction would be that a good number of random alleles of npr1 would be RIM+ and partially receptive to SA. We previously showed that for SA perception, there are genetic interactions between the npr1 alleles and the NPR1 paralogs [11]. The work reported herein points to a genetic interaction too, this being between npr1 alleles on one side and the BOPs on the other. Thus, the RIM- alleles were a phenocopy of the bop1 bop2 mutant in defense but not in development. This discrimination was a consequence of the different thresholds for the phenotype in development and defense (Figure 5b,c).
Mechanistically, the levels of expression of the BOPs were low in comparison to NPR1 (Additional file 1), so a direct or indirect negative interference of NPR1 with the BOPs would be favored stoichiometrically. Once the pathogen was inoculated, the levels of SA would rise and in a wild type plant NPR1 is degraded as part of the signaling process [48]. In an npr1 background, this signaling would not be transmitted and perhaps the npr1 proteins would be able to interfere longer in RIM. This would explain the behavior of NPR1-HBD in npr1-1 (v 2b); NPR1-HBD in the cytoplasm did not complement npr1-1 in the RIM phenotype, but when DEX was applied there was complementation of the phenotype. Likely, when no DEX was present npr1-1 would somehow interfere with the function of the BOPs. When DEX was present, the presence of NPR1-HBD in the nucleus would trigger the degradation of both NPR1-HBD and npr1-1. If npr1-1 was degraded, the BOPs would function normally.
There was no evidence for a direct interaction in yeast, since the presence of NPR1 or mutated versions of this protein did not interfere in the interaction between BOP1 and BOP2 in a consistent manner with the phenotype (Figure 8a). A first alternative was that the interference of the RIM- alleles would occur with the BOPs without affecting the interaction between the BOPs. A second alternative would be that the RIM- alleles would interfere with other proteins that normally interact with the BOPs. In both cases there is a family of proteins that interacts with both NPR1 and the BOPs, the transcription factors TGAs [49], with -again- functional redundancy (Figure 8b). Two out of three RIM- alleles enhanced or stabilized the BOPs-TGAs interaction, while two out of three RIM+ alleles did not (Figure 8c and data not shown). It was clear that the npr1 mutated proteins had an unpredicted effect on the BOPs-TGAs interaction, but the yeast experiments did not produce an absolute answer about the role of npr1 proteins in RIM. We speculate that in planta, all the RIM- alleles enhance the interaction between the BOPs and the TGAs, titering out the TGAs and thus rendering them unable to fulfill their function of triggering defense. On the other hand, the RIM+ alleles (including the null alleles), and NPR1 would not affect the interaction either way. Since in the yeast assays two out of three alleles worked as proposed in either way, it may be possible that a factor(s) is present in the plant that is not in yeast, or perhaps the fact that there are ten TGAs[49], and that NPR1 is expressed between 3 and 18 times more than BOP1 + BOP2 (Additional file 1) could explain this difference. If this hypothesis were to be true, it will definitively explain the role of npr1 in RIM.
In sum, we have shown that, in wild type conditions, the BOPs and the TGAs (but not NPR1) are required for the resistance triggered by methyl jasmonate against Pto. We propose that the phenotype of the npr1 RIM- alleles is caused by their interference between BOPs and TGAs.
Plant growth and inoculation
Arabidopsis thaliana (L.) Heynh. was sown and grown as described [23] in controlled environment rooms with days of 8 h at 21°C, 150 μmol m-2 s-1, and nights of 16 h at 19°C. Treatments, inoculations, and sampling started 30 minutes after the initiation of the artificial day to ensure reproducibility. The following genotypes were used: npr1-1 and npr1-3 [27]; npr2, npr1-20 to npr1-71, and combinations of npr1-70 with other genotypes [11]; 35SCaMVp:NPR1HBD[15]; sid2[30]; eds5[29]; NahG[28]; npr3 and npr4[7]; bop1-3 and bop2-1 [21]; coi1-40 (Dobón, Wulff, Canet and Tornero, to be published elsewhere); kant6, pan1-1 to pan1-3, tga1, and tga7[50]; 35S:BOP1 and 35S:BOP2[33]; etr1-3 [39]; tga2 tga5 tga6[41]. Pseudomonas syringae pv. tomato DC3000 (Pto) was grown, inoculated and measured as described [51]. Briefly, plants of 14 days were inoculated by spray with Pto at OD600=0.1 with 0.02% Silwet L-77 (Crompton Europe Ltd, Evesham, UK). Three days later, the amount of colony forming units (cfu) per plant was quantified and represented in a logarithmic scale. When indicated, a strain of Pto lacking coronatine was used (Pto(cfa–),[37]). For all the experiments, at least three independent treatments were performed (three independent sets of plants sown and treated on different dates).
Expression in planta and in yeast
NPR1 and six alleles of this gene were cloned in pDONR222 or pDONR221 (Invitrogen, Barcelona, Spain) and then transferred to pMDC43 [52] for expression in planta with GFP and to pARC352 [53] for expression in yeast. Similarly, BOP1, BOP2, TGA2, TGA5, and TGA6, were cloned and then transferred to pDEST22 and pDEST32 (Invitrogen) for expression in yeast. Yeast n-hybrid analyses were done as described [54], and the interactions were quantified as described [55]. N. benthamiana leaf tissue was mounted in water under a coverslip 4 days after infiltration with Agrobacterium tumefaciens containing the constructs. All imaging was conducted with a Leica TCS SL confocal laser scanning microscope (Leica, Barcelona, Spain) using an HCX PL APO CS 40X/1.25 oil objective to study the subcellular localization of the fluorescence-tagged proteins. Green fluorescent protein was visualized by 488-nm excitation with an Ar laser, and its emissions were examined with a band-pass filter for 500 to 530 nm. The primers used are included as Additional file 1. Primers and chemical products were purchased from SIGMA (St. Louis, MO, USA) unless otherwise is stated. For the construction of amiRNA(BOP1-BOP2), the plasmid pRS300 was modified [32], cloned in pGW14 [56], and plants were transformed as described [57].
Chemical treatments
To measure the effect in Pto growth 100 μM methyl jasmonate (MeJA) in 0.1% DMSO and 0.02% Silwet L-77 (Crompton Europe Ltd) was applied by spray one day before the pathogen inoculation [25]. Dexamethasone was applied at 2 μM diluted in water from a stock of 20 mM in EtOH. 1-Aminocyclo- propane-1-carboxylic acid (ACC) was sprayed at 1 mM in water with 0.02% Silwet L-77.
In vitro growth
For in vitro culture, plants were grown in Johnson's media [58] with 1 mM KH2PO4. When indicated, the plates were supplemented with 50 μM MeJA. The length of the roots was measured with ImageJ software [59]. Senescence induced by MeJA was measured as described [36].
Total RNA from 3-week-old (Figure 5c) or 6-week-old plants (Figure 6b) was extracted with Trizol (Invitrogen), following the manufacturer's instructions. cDNA was synthesized with RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, Madrid, Spain), and the quantitative PCR performed with LuminoCt Sybr Green qPCR Ready Mix (SIGMA) in a 7000 RT-PCR Systems machine (Applied Biosystems, Madrid, Spain), following the manufacturer's instructions. For each measurement three biological replicates were done. The obtained values were referred to the geometric average of three reference genes (At3G18780, At1G49240, and At5G60390), as described [60], and normalized, being the value of Col-0 in mock equal to one. The list of primers used is provided in Additional file 1.
Competing interests
The authors declare that they do not have any competing interest.
Authors' contributions
JVC, AD, and JF performed the experiments, analyzed the data and revised the paper. PT designed the research, analyzed the data and wrote the paper. All authors read and approved the final manuscript.
This work was supported by the "Ministerio de Economía y Competitividad" (MINECO) of Spain (grant BIO201018896 to PT, a JAE-CSIC Fellowship to JVC and a FPI-MINECO to AD) and "Generalitat Valenciana" of Spain (grant ACOMP/2012/105 to PT). Thanks to Dr. Xinnian Dong for NPR1 overexpression lines and to Dr. Ove Nilsson for BOPs overexpression lines. We appreciate the opinions and generous help of Drs. Vicente Ramirez, Pablo Vera, and Shelley Hepworth about the manuscript.
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Plant Cell 2002, 14(1):275-286. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Wildermuth MC, Dewdney J, Wu G, Ausubel FM: Isochorismate synthase is required to synthesize salicylic acid for plant defence.
Nature 2001, 414(6863):562-565. PubMed Abstract | Publisher Full Text
Khan M, Xu M, Murmu J, Tabb P, Liu Y, Storey K, McKim SM, Douglas CJ, Hepworth SR: Antagonistic interaction of BLADE-ON-PETIOLE1 and 2 with BREVIPEDICELLUS and PENNYWISE regulates Arabidopsis inflorescence architecture.
Plant Physiol 2011, 158(2):946-960. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D: Highly specific gene silencing by artificial microRNAs in Arabidopsis.
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Dombrecht B, Xue GP, Sprague SJ, Kirkegaard JA, Ross JJ, Reid JB, Fitt GP, Sewelam N, Schenk PM, Manners JM, et al.: MYC2 Differentially Modulates Diverse Jasmonate-Dependent Functions in Arabidopsis.
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He Y, Fukushige H, Hildebrand DF, Gan S: Evidence supporting a role of jasmonic acid in Arabidopsis leaf senescence.
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Ton J, Mauch-Mani B: Beta-amino-butyric acid-induced resistance against necrotrophic pathogens is based on ABA-dependent priming for callose.
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Chang C, Kwok SF, Bleecker AB, Meyerowitz EM: Arabidopsis ethylene-response gene ETR1: Similarity of product to two-component regulators.
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Jun JH, Ha CM, Fletcher JC: BLADE-ON-PETIOLE1 coordinates organ determinacy and axial polarity in arabidopsis by directly activating ASYMMETRIC LEAVES2.
Plant Cell 2010, 22(1):62-76. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Zhang Y, Tessaro MJ, Lassner M, Li X: Knockout analysis of Arabidopsis transcription factors TGA2, TGA5, and TGA6 reveals their redundant and essential roles in systemic acquired resistance.
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Delaney TP, Friedrich L, Ryals JA: Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance.
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Ha CM, Jun JH, Nam HG, Fletcher JC: BLADE-ON-PETIOLE 1 and 2 control Arabidopsis lateral organ fate through regulation of LOB domain and adaxial-abaxial polarity genes.
Plant Cell 2007, 19(6):1809-1825. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Xu M, Hu T, McKim SM, Murmu J, Haughn GW, Hepworth SR: Arabidopsis BLADE-ON-PETIOLE1 and 2 promote floral meristem fate and determinacy in a previously undefined pathway targeting APETALA1 and AGAMOUS-LIKE24.
Plant J 2010, 63(6):974-989. PubMed Abstract | Publisher Full Text
McKim SM, Stenvik GE, Butenko MA, Kristiansen W, Cho SK, Hepworth SR, Aalen RB, Haughn GW: The BLADE-ON-PETIOLE genes are essential for abscission zone formation in Arabidopsis.
Development 2008, 135(8):1537-1546. PubMed Abstract | Publisher Full Text
Ha CM, Jun JH, Fletcher JC: Control of Arabidopsis leaf morphogenesis through regulation of the YABBY and KNOX families of transcription factors.
Genetics 2010, 186(1):197-206. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Stein E, Molitor A, Kogel KH, Waller F: Systemic resistance in Arabidopsis conferred by the mycorrhizal fungus Piriformospora indica requires jasmonic acid signaling and the cytoplasmic function of NPR1.
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Spoel SH, Mou Z, Tada Y, Spivey NW, Genschik P, Dong X: Proteasome-mediated turnover of the transcription coactivator NPR1 plays dual roles in regulating plant immunity.
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Jakoby M, Weisshaar B, Droge-Laser W, Vicente-Carbajosa J, Tiedemann J, Kroj T, Parcy F: bZIP transcription factors in Arabidopsis.
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Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, et al.: Genome-wide insertional mutagenesis of Arabidopsis thaliana.
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Tornero P, Dangl JL: A high throughput method for quantifying growth of phytopathogenic bacteria in Arabidopsis thaliana.
Plant J 2001, 28(4):475-481. PubMed Abstract | Publisher Full Text
Curtis MD, Grossniklaus U: A gateway cloning vector set for high-throughput functional analysis of genes in planta.
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Ciannamea S, Kaufmann K, Frau M, Tonaco IA, Petersen K, Nielsen KK, Angenent GC, Immink RG: Protein interactions of MADS box transcription factors involved in flowering in Lolium perenne.
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Vidal M, Legrain P: Yeast forward and reverse 'n'-hybrid systems.
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Clough SJ, Bent AF: Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.
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Johnson CM, Stout PR, Broyer TC, Carlton AB: Comparative chlorine requirements of different plant species.
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Abramoff MD, Magalhaes PJ, Ram SJ: Image Processing with ImageJ.
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Genome Biol 2002., 3(7)


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Faculty Publications 2003
Blackwell, Peggy J., Mary H. Futrell and David G. Imig. "Burnt Water Paradoxes of Schools of Education." Phi Delta Kappan. 84(5):365-361. 2003.
Boulmetis, John. SEE: Harlow, Gary.
Brand, Stephen. SEE also: Information Works!
Brand, Stephen, Robert Felner, Minsuk Shim, Anne Seitsinger and Thaddeus Dumas. "Middle School Improvement and Reform: Development and Validation of a School-Level Assessment of Climate, Cultural Pluralism, and School Safety." Journal of Educational Psychology. 95(3):570-588. September 2003.
Burden, Paul R. and David M. Byrd. Methods for Effective Teaching. 3rd ed. Allyn and Bacon. 2003. 412p.
Byrd, David M. SEE: Burden, Paul R.
Dash, Gordon H., Jr., Choudary R. Hanumara and Nina Kajiji. "Neural Network Architecture for Modeling FX Futures Options Volatility." IN: 2003 Northeast Decision Sciences Conference Proceedings. 2003.
Dash, Gordon H., Jr. and Nina Kajiji. "New Evidence on the Predictability of South African FX Volatility in Heterogenous Bi-Lateral Markets." African Finance. 5(1). 2003.
Deeney, Theresa. SEE also: Wolfe, M.
Deeney, Theresa, Editorial Research Board. Reading Online. Vol.7, 2003.
Downing, June E. and Joanne Eichinger. "Creating Learning Opportunities for Students With Severe Disabilities in Inclusive Classrooms." Teaching Exceptional Children. 36(1):26-31. September/October 2003.
DuBois, David L., Robert D. Felner, Erika M. Lockerd, Gilbert R. Parra and Christy Lopez. "The Quadripartite Model Revisited: Promoting Positive Mental Health in Children and Adolescents." IN: Cognitive Therapy WIth Children and Adolescents: A Casebook for Clinical Practice. 2d ed. Edited by Mark A. Reinecke, Frank M. Dattilio et al. Guilford Press. 2003. p.402-433.
Dumas, Thaddeus. SEE: Brand, Stephen.
Dutwin, Phyllis, Carol Altreuter and Kathleen A. Guglielmi. Test of Adult Basic Education: The First Step to Lifelong Success. McGraw-Hill. 2003.
Eichinger, Joanne. SEE: Downing, June E.
Felner, Robert D. SEE: Brand, Stephen; DuBois, David L.; Information Works! Shim, Eunjae.
Guglielmi, Kathleen A. SEE: Dutwin, Phyllis.
Harlow, Gary, John Boulmetis, Phillip G. Clarke and George H. Willis. "Computer-Assisted Life Stories." Computers in Human Behavior. 19(4):391-406. July 2003.
Imig, David G. SEE: Blackwell, Peggy J.
Information Works! Measuring Rhode Island Schools for Change, R. D. Felner, E. Krieger, N. Kajiji, S. Brand, L. Kellog, J. Shim, M. Shim and J. Steiny. Department of Education, University of Rhode Island, National Center on Public Education and Social Policy. 2003.
Kajiji, Nina. SEE: Dash, Gordon H., Jr.; Information Works!
Marsh, Colin J. and George Willis. Curriculum: Alternative Approaches, Ongoing Issues. 3rd ed. Merrill/Prentice Hall. 2003. 389p.
McKinney, W. L. "Book Review: De los Reyes, Eileen. Pockets of Hope: How Students and Teachers Change the World." Choice. February 2003.
Seitsinger, Anne. SEE: Brand, Stephen; Information Works!
Shim, Eunjae, Minsuk K. Shim and Robert D. Felner. Automation of Survey Data Processing, Documentation and Dissemination: An Application to Large-Scale Self-Reported Educational Survey. (ERIC ED 466 692) February 2003.
Shim, Minsuk K. SEE: Brand, Stephen; Information Works! Shim, Eunjae.
Willis, George H. SEE: Harlow, Gary; Marsh, Colin J.
Wolf, M. and T. Deeney. "Dyslexia." IN: International Encyclopedia of Linguistics. 2d ed. Oxford. 2003. p.472-475.
Return to Faculty Publications
2003 Table of Contents.
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text: Now online shopping is not just limited to the virtual shops. On Indiatimes shopping, you can buy discount vouchers and gift cards for great shopping brands like Big Bazaar, Shoppers stop, Lifestyle, mothercare, Central, Puma, van Heusen, Allen Solly, Surat Diamonds, Landmark and many more which will offer you great deals and discounts on your daily shopping. You can also buy eatery deals and discounts with café moments cards, dominos Pizza vouchers and much more. Shop online, shop offline and shop till you drop with great deals and discounts.


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Available to faculty, administrators and staff, the Office of Technology Management provides customized technology training solutions, both for groups and individuals.
We also offer step-by-step tutorials on the web which can be viewed in our Wiki.
If you need training on using MySuffolk, RedDot Content Management System, or Datatel Colleague please visit the Human Resources training section.


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Caterham are easily on the level of say Tyrrell, Arrows and Minardi in the mid-90s. Therefore, there's something not quite right with F1 if this team can't even have a CHANCE of scoring points.
(I think it's the super-reliability and even worse, no gravel traps so the drivers now get away with murder and simply rejoin the track, whereas it would be game over in the past)
With the amount of criticism they, Marussia and HRT get on here, you'd think they were talking about Eurobrun, Coloni and Pacific...
Kovalainen deserves to be fighting for points, podiums and wins. I hope Caterham can succeed without him, cos they certainly look like they're getting the better end of the deal. What about Adrian Sutil if Massa's not interested?


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How Algae 'Enslavement' Threatens Freshwater Bodies
Jerusalem, Israel (SPX) Aug 19, 2010
How toxic, blue-green algae out-compete other organisms through a form of selfish "enslavement" - and by so doing proliferate dangerously in freshwater bodies - has been described by a researcher at the Hebrew University of Jerusalem.
In general, the increasing occurrence of toxic cyanobacterial (blue-green algae) blooms in freshwater bodies is a matter of growing international concern due to their detrimental impact on drinking water quality and, in extreme cases, causing death to humans, livestock and wild animals.
Thus, the new Hebrew University research can be of value to water authorities seeking ways to control this algae infiltration.
A toxic blue-green alga known as Aphanizomenon ovalisporum was first detected in Lake Kinneret (Sea of Galilee) in 1994, and its presence has been noted each summer thereafter. The conditions promoting these toxic blooms and other toxin formations in freshwater bodies were not known.
However, now in a paper to be published online on Aug. 12 in Current Biology, Yehonatan Bar-Yosef, a Ph.D. student in Prof.
Aaron Kaplan's group at the Alexander Silberman Institute of Life Sciences at the Hebrew University of Jerusalem, has suggested a novel mechanism to explain the ability of Aphanizomenon to form massive toxic blooms by overcoming competition from other microorganisms in the water. (Kaplan is the Bernice and Aaron Beare Family Professor of Environmental Plant Physiology.)
Aphanizomenon is known to produce the toxin cylindrospermopsin (CYN). Secretion of the CYN, Bar-Yosef found, induces phosphate-limitation responses in other microorganisms in the ecosystem, even in the presence of ample phosphate in the water. The phosphate mineral is an essential nutrient for growth in many organisms.
The result is that Aphanizomenon is able to attain a relative advantage in phosphate-absorption capability, thus gaining dominance in the competition for nutrients.
The investigators have used the term "enslavement" to describe this novel interspecies interaction, mediated by CYN. This research provides an explanation for the significant rise in massive cyanobacterial bloom events worldwide during the last decade despite attempts of water management authorities to reduce the inflow of nutrients, especially phosphate, entering from watersheds.
The research on the Kinneret water was carried out close cooperation with Dr. Assaf Sukenik and Dr. Ora Hadas from the Kinneret laboratory of the Israel Institute of Limnology and Oceanography.
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Water News - Science, Technology and Politics
Tokyo (AFP) Aug 18, 2010
Japanese trading giant Itochu Corp. will start selling water-processing equipment in China to help combat the country's serious problems with water pollution, a company spokesman said Wednesday. ITC Green and Water Corp., a water-processing unit of the Tokyo-based trading house, will later this year market a new water-treatment system developed by Japanese contractor Matsue Doken, the spokes ... read more
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Silvio Berlusconi Holds Political Rally In Milan
Supporters hold flags of the Popolo della Liberta and Lega North... News PhotoDemocracy,Election,Flag,Holding,Horizontal,Italy,Milan,People of Freedom - Italian party,Political Party,Political Rally,Politics,SupporterPhotographer Pier Marco TaccaCollection: Getty Images News 2013 Pier Marco TaccaMILAN, ITALY - FEBRUARY 18: Supporters hold flags of the Popolo della Liberta and Lega North parties during a political rally on February 18, 2013 in Milan, Italy. Berlusconi is entering the last week of campaigning for his party Popolo della Liberta. Italians go to the polls February 24-25. (Photo by Pier Marco Tacca/Getty Images)


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Film Screening: The Great Gatsby
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Director Baz Luhrmann's blockbuster adaptation of the novel by F. Scott Fitzgerald, screened outdoors on the East Campus Quad. Co-sponsored by the East Campus Libraries and Devils After Dark.
Movie/Film, Orientation, Social, and Alumni/Reunion


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The 1972 Miami Dolphins are the NFL benchmark for success and a great source of pride for all Dolfans. In the 35+ years since the perfect season no other team has been able successfully run the table and join the 72 Dolphins as undefeated. Several have come close, most notably the 85 Bears and the 07 Patriots but neither team was able to achieve perfection as the Dolphins had done.
After being humiliated by the Dallas Cowboys 24-3 in the Super Bowl the Miami Dolphins entered the 72 season with a chip on their shoulder; they had something to prove. They didn't set out to go undefeated; they just played every game with intensity and everything fell into place. Players had no idea of the enormity of the perfect season they just wanted to get back to the big game and win it.
The 72 team was built on a power running game led by Larry Csonka and Mercury Morris and a stout defense made up of "no namers" who excelled as a unit. This formula would prove important as starting Quarterback, Bob Griese, would go down in the 5th game with a broken leg. Earl Morrall, a 38 year old backup, substituted for Griese leading the team into the playoffs while not losing a single game. Griese returned for his first start to defeat the Redskins in the Super Bowl.
Shula, in reflecting on the championship game that year, said that when the team was up over the Redskins 14–0 he had thoughts of finishing the game 17-0 , matching what would be their record, when he decided to kick the field goal which became one of the more infamous plays in Dolphin history. The field goal was blocked and rather than simply fall on it kicker, Garo "I kick a touchdown" Yepremian, picked up the ball and tried to pass it down field. The ball slipped out of his hand and was batted into the arms of Redskins cornerback Mike Bass,who ran the ball back 49 yards, cutting the lead in half.
Luckily for Yepremian the Dolphins were able to hang on and defeat the Washington Redskin 14-7 to preserve their undefeated season by winning the Super Bowl.
Over the years many have argued that the 72 Dolphins were not the greatest NFL team ever and on paper they may not have been. Even so, no other team since has accomplished what the 72 Dolphins did in winning every game. As Csonka once said, "Perfection ends a lot of arguments." One day a team may indeed join them in "Perfectville" but they will always be the first.
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About Scituate Rod & Gun Club
The Scituate Rod & Gun Club, incorporated in 1929, is one of the oldest clubs on the South Shore.
We are situated on 29.5 acres on the West side of Route 3A in North Scituate, about 3/10 mile South of the Cohasset line. An active land acquisition program effectively doubled the size of the Club during 1994.
Although only a small Club, SRGC offers something for just about every taste in shooting sports with a calendar of events scheduled throughout the year.
Events include:
- Skeet on Sunday afternoons and Wednesday evenings.
- Trap on the 2nd and 4th Saturdays.
- SASS (Single Action Shooting Society) matches throughout the year.
- Pistol matches.
- Rifle and Muzzleloader matches.
- A Turkey Shoot in the Fall.
- A Kid's Fishing Derby in May. 2014 Fishing Derby and
2014 Vocational Life Skills Fishing Derby
- For the archers, a 3-D course winds its way around our property with practice butts available behind the Clubhouse.
- Activities listed above in grey are on hold pending range improvements.
Adjacent to the Clubhouse is a small pond for members to use. The pond is a favorite with kids in the summer while Dad, and sometimes Mom too, play with their toys.
The Club holds two business meetings per month; on the first and third Mondays starting at 8:00 pm. Prospective new members are voted in at these meetings.


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By the OGJ Online Staff
HOUSTON, Jan. 8 -- Holly Corp. subsidiary Navajo Refining Co. LP has received permits needed to install a gas oil hydrotreating unit at its 60,000 b/d refinery at Artesia, NM, and expand refining capacity.
The cost of the hydrotreater project will be reduced by using hydrotreating equipment purchased from an Illinois refinery, said Holly, which estimates the project will cost $48 million, including $15 million already spent on engineering and the purchase and relocation of equipment.
The hydrotreater installation, to be completed in 2003, will allow Navajo to expand its ability to produce California-grade gasolines and gasolines that will meet sulfur reduction requirements before regulatory deadlines, said Holly, which did not reveal the target production capacity.
Navajo also received permission to expand refining capacity to 70,000 b/d, expected by yearend 2003. The permits will also permit a second-phase expansion of crude capacity, up to 80,000 b/d, but no schedule for the further expansion has been set.
Holly will need to obtain additional permits to implement needed modifications at Navajo's Lovington, NM, refinery operated in conjunction with the Artesia facility. Those modifications should also be complete by the end of 2003.
Matthew Clifton, Holly president, said, "We are extremely pleased to produce these cleaner-burning gasolines for our New Mexico, Arizona, Texas, Colorado, and Utah customers ahead of our regulatory deadlines."
Last month, Holly agreed to a settlement of alleged federal clean air violations with the US Department of Justice and the Environmental Protection Agency that required it to spend $16-21 million to control emissions from its two refineries (OGJ Online, Dec. 20, 2001).


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SICPA Holdings
Child Organization: SICPA Product Security
Figures are based on lobbying activity reported to the Senate Office of Public Records. Reported dollar amounts are required to be accurate only to the nearest $20,000. For organizations whose primary business is lobbying, we display total income and top clients. For organizations that are not primarily lobbying firms, we display total amount spent on lobbying and top lobbying firms hired.
For more information, please see our lobbying methodology page.
Lobbying on Behalf of SICPA Holdings
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Lauren Merkin
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Description: Large clutch. Serpent embossed lambskin clutch with paneled leather detail. Hinged frame closure. Signature striped shimmer interior lining with large zip pocket and metal embossed logo plate. Made in NYC. Body length 14½ inches; height 7½ inches; width 1 inch.
Material: Serpent Embossed Lambskin
Brand: Lauren Merkin
Origin: United States


text: American College of Surgeons Education Patients and Family The Recovery Room Recovery Room Episodes Cesareans, Foreign Medical Graduates Division of Education About the Division of Education Contact Us Courses Courses Overview ACS Comprehensive General Surgery Review Residents as Teachers and Leaders Surgeons as Educators Surgeons as Leaders ACS Education and Training Publications Publications Overview Selected Readings in General Surgery ACS Multimedia Atlas of Surgery Volumes Ethical Issues in Clinical Surgery Horizon Scanning in Surgery Successfully Navigating the First Year of Surgical Residency Issues in Surgical Ethics ACS Video Library MyCME and Specialty Certificates About CME Claim and Manage Your CME Claim Clinical Congress CME CME Calendar Find CME Opportunities JACS CME Accreditation, Verification, and Validation Accreditation, Verification, and Validation Programs Accredited Education Institutes Accredited Education Institutes AEI Calendar Maintaining AEI Accreditation AEI Consortium Annual AEI Consortium Meeting Annual ACS-AEI Postgraduate Course AEI News Resources for AEIs Contact the AEI Staff CME Accreditation CME Joint Providership Program CME Joint Providership Program Prior to Your CME Meeting During and After Your Joint Provided Meeting Contact Us Program for Validation and Verification of Surgical Knowledge and Skills Program for Validation and Verification of Surgical Knowledge and Skills About Contact Us Requirements National Ultrasound Faculty Resources Educational Resources E-Learning E-Learning Resource Center Practice Management Online Course Clinical Congress 2015 Webcast Sessions Frequently Asked Questions Evidence-Based Decisions in Surgery Medical Student Resources So You Want to Be a Surgeon So You Want to Be a Surgeon Your Residency Search Section I: Surgical Traits Section II: How to Find a Surgery Residency Section III: Surgical Specialties Residency Search Links of Interest Residency Assist Page Resident Resources Teaching Resources Online Guide to Choosing a Surgical Residency Online Guide to Choosing a Surgical Residency Overview Choosing Your Surgical Residency Advisor Medical Students FAQs Value of Electives During Medical School What to Expect in a Surgical Residency Program Information for Medical Students Surgical Career Lifestyle Issues ACS Programs for Medical Students and Residents Websites for Prospective Surgical Residents Post-Residency Fellowships Suggested Readings for Residents Choosing a Residency Surgical Interest Group Medical School Timeline Clinical Congress Clinical Congress Overview Welcome Scientific Program Special Interest Sessions CME Credit Registration and Program Planner Media Information Search for Sessions Housing and Travel Future Clinical Congresses Clinical Congress 2014 Clinical Congress 2014 Program 2014 Clinical Congress News Patients and Family Patient Education Patients Patient Resources Operation Brochures for Patients Prepare for Your Operation Skills Programs Informed Consent What is Correct-Site Surgery? 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Over 30 percent of American births are currently done by c-section. And it's a number that isn't likely to drop any time soon. Once a woman gets a cesarean, she'll almost always have another when she gives birth again. Recent revisions to guidelines for VBACs – or "vaginal birth after cesarean" – are intended to reduce multiple c-sections. On today's show, why those guideline revisions may have less impact than intended and just how concerned we should be about the rising cesarean rate. And later in the show, how foreign medical grads are contributing to U.S. health care. Listen to this episode. Segment 1: Cesareans (00:00-13:40) Guest: Dr. Bill Grobman, co-author of revised VBAC ("vaginal birth after cesarean") guidelines for College of Obstetricians and Gynecologists Since 1970, the cesarean delivery rate in the U.S. has jumped from 5 percent to more than 30 percent. (The rate recommended by the World Health Organization is 15 percent.) A lot of factors have contributed to the increase: more women are giving birth at an advanced age and more are having multiple births - twins and triplets - some as a result of fertility treatments. Doctors' fears of litigation have contributed, as well; as could have the fact that cesareans pay doctors more - on average, almost twice as more - as a vaginal birth. Another factor: electronic fetal monitoring. Introduced in the '70s, it's now become standard in most births. Changes in the baby's heart rate - a possible sign of too little oxygen - will often result in a c-section; but there's little proof that the test actually saves lives. And despite the fact that most women who've had a c-section can safely give birth vaginally, less than 10 percent are having VBACs - or "vaginal birth after cesarean." VBAC rates have fluctuated over the years largely in response to shifting guidelines. A 1999 revision stated that hospitals had to have surgery and anesthesia teams "immediately available" - that is, on site - in the case of a woman requesting a VBAC. The costs of this recommendation lead many hospitals - especially smaller and rural hospitals - to ban the practice. Just last month, the college of Obstetricians & Gynecologists revised its guidelines again. The new guidelines reaffirm the safety of VBAC for most women. But they also retain the "immediately available" language regarding emergency care. A recommendation that women be allowed to accept "increased levels of risk" has also been added. The question remains whether this will have the hoped-for outcome - that is, an increase in VBACs and a decrease in the overall cesarean numbers. Segment 2: Foreign Medical Graduates (13:43-28:00) Guest: James Hallock, former president of the Educational Commission for Foreign Medical Graduates One of the biggest problems facing the health care industry today is a shortage of primary-care physicians. As U.S. med school grads choose specialization over general medicine, foreign medical graduates are coming to the U.S. for residency programs, and many are sticking around and helping to fill primary-care vacancies. By a wide margin, the largest number of foreign medical graduates have come from India, but significant numbers have also come from Pakistan, the Philippines, China, Egypt, and the Dominican Republic. But how comparable is medical education in these countries? And how equipped are these international doctors to care for sick Americans? Since 1956, the Educational Commission for Foreign Medical Graduates has evaluated and certified foreign medical grads for U.S. residency programs. In 2008, the commission issued over 10,000 certificates. If the commission's previous statistics apply, about 7,000 will end up practicing medicine here in the States.