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Tuesday, November 15, 2011 More items for Program/Project Management
An environmentally sensitive segment of the proposed Keystone XL pipeline will be rerouted and a final decision on the controversial $7 billion project delayed until after the 2012 U.S. election, officials have announced.
The White House said Thursday (Nov. 10) that it would seek to shift the 1,700-mile pipeline away from the most sensitive areas.
State Department
That work, and subsequent review, will require a delay of at least a year, the Obama Administration said.
TransCanada Corp., the pipeline developer, said later that it would reroute the line.
'Open, Transparent Process'
"Because this permit decision could affect the health and safety of the American people as well as the environment, and because a number of concerns have been raised through a public process, we should take the time to ensure that all questions are properly addressed and all the potential impacts are properly understood," President Obama said in a statement.
"The final decision should be guided by an open, transparent process that is informed by the best available science and the voices of the American people."
Environmental Impact Studied
The decision slapped a surprising halt on a project that had won the State Department's favor in August. At the time, the department released a nine-volume, 1,000-page Final Environmental Impact Statement (FEIS) showing minimal environmental risk from the project.
The FEIS was the State Department's third, and final, review since TransCanada proposed the pipeline in 2008.
Although the EIS did not constitute a final decision—one was not expected until the end of the year—it was considered to have cleared a major hurdle from the project.
The study enraged environmental groups, who continued to press opposition to the plan in rallies and demonstrations, culminating in a march Nov. 6 that drew thousands of protesters to the White House.
Later that week, the State Department said it would seek an alternative route for a small portion of the pipeline that runs through an environmentally sensitive part of Nebraska known as the Sand Hills.
That will require a new environmental review that will take until at least the first quarter of 2013, the department said.
Decision Hailed, Reviled
Environmentalists called the new delay a clear victory, while industry and labor assailed the decision, saying the project would have created jobs and increased U.S. oil security.
"If Keystone XL dies, Americans will still wake up the next morning and continue to import 10 million barrels of oil from repressive nations, without the benefit of thousands of jobs and long-term energy security," said TransCanada Chief Executive Russ Girling.
Jack Gerard, director of the American Petroleum Institute, said the pipeline delay was "clearly about politics and keeping a radical constituency opposed to any and all oil and gas development in the president's camp for 2012."
Tar Sands Oil
The pipeline would carry up to 830,000 barrels of heavy crude oil per day and was expected to begin operation in 2013, if construction had begun in 2012, the company says. The crude would be extracted from the oil sands areas in Alberta, Canada. Oil sands (also known as tar sands) are a combination of clay, sand, water, and bitumen.
Critics note that TransCanada's current 1,300-mile Keystone system has sustained 14 spills since it began operation in June 2010, sparking intense debate over whether the company could safely operate the so-called Keystone XL project, which would nearly double the system's capacity.
The XL project—officially, the Keystone Gulf Coast Expansion Project—would be the third phase of the $13 billion Keystone pipeline network.
TransCanada said it believed the pipeline would eventually be built.
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obama won the nobel peace prize??? | |
Anonymous Coward User ID: 17917453 Australia 07/03/2012 07:40 AM Report Abusive Post Report Copyright Violation | |
Anonymous Coward User ID: 19045692 United States 07/03/2012 10:09 AM Report Abusive Post Report Copyright Violation | He was AWARDED it... he DIDN'T win it. The "prize" was provided to him from the controlling / decieving evil money mongrols of the world in order to prop the anti-god Obama in the public eye. Yessss, the irrelevent MSM continues to create good to be evil and evil to be good. I encourage ea of you to begin to think critically and not led into an eternity of damnation. We all make choices. I pray that you will choose wisdom as your choice. |

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Jan 27, 2013
My wife an me fucked every day some times or then 2 time a day then on our 7th year it was dry to where we did it 1time a week then 1 time a month then one day it was like any other day love u kiss then she went to work I didn't see her for a day an a half I know where she went but I played dumb to see what she would tell me said she was at a friends I said OK she went to take a bath I was pissed so I went to tell her I know what she did she said OK I did I fucked my ex last night
Since: Jul 12 533 Perth, Australia |
Jan 29, 2013
that is so hot, I know the thought of being with another guy turns my wife on,but it has been the stupid things I have done to set her up and just pressuring her to do it which has made her go against actually doing it. |
Since: Jul 12 533 Perth, Australia |
Jan 29, 2013
How did you start sharing her mathisha? |
Since: Apr 12 98 |
Jan 29, 2013
Wanna talk off of here |
Since: Jan 13 932 |
Jan 29, 2013
I agree with you 100%. Nobody outside of the other men involved ever knew about or suspected what we were doing behind closed doors except our swap couple and the men in our MFM's. My wife was always treated with the utmost respect. We still love and respect each other. There were never any emotional hang-ups or marriages broken up. Several of the men were married friends of ours who's wives never knew. I just with my wife's sexual appetite had not diminished after 17 or 18 years. I would love to stil be having them. |
Since: Jul 12 533 Australia |
Jan 30, 2013
that would be hard to take,if you don't mind me asking what on earth did you have a disagreement about that? Surely in the right situation she would give it a go |
United States |
Jan 31, 2013
A husband that shares his wife gets better lovin than any other-I can attest to this, we love the lifestyle, never going back that's for sure-been doing this for about a decade n we both love it! |
Feb 1, 2013
I got up off of her he came around an I was coming out of her he said damn dude then pushed his cock in to her an was going at it hard I was watch an I was getting hard again he was giving it his all but she just played there with her legs up but not like she had them with me she let out a couple of little mones I was hard again an I said get on to an ride him while she was riding him well u know what I did with mine all together it was about 2 hours then he left an she said can we do it again I cheated cause u never had time for me but after tonight it just u an me cause I'm never going to find a Guy like u or have one a big as u I don't know what I was thinking I asked her how mine time did u cum with him she said 3 then I said what about with me she said 26
Feb 1, 2013
A month later she told me she told her girlfriends what I let her do I said why don't tell them that said one of my friends thinks your hot an wants to try it with I said OK she came over that night her an my girl started making out to turn me on cause she told her girlfriend I have been wanting her for a long time since I seen her in our pool 2 summers ago an told her Id like to see them together her girlfriend started going down on her man I was hard then my girl went do on her then they both went down on me then I put both of them on there backs an did both of them then pounded both of the so mine def was we all loved it so much now we do it once a month an I'm the only Guy aloud.
Feb 1, 2013
I do I love get gfs wet |
Since: Apr 12 199 Location hidden |
Feb 1, 2013
Are you a graduate of elementary school? Or still in the 3rd grade? |
Since: Jul 12 533 Perth, Australia |
Feb 3, 2013
I hope you get to share her Jerry, then you can tell us what it is like to watch another man enter your wife. |
Feb 3, 2013
MikeIrons - that was a very disparaging comment. Normally I would be proud of you, but your second sentence doesn't have a subject, so I have to give you a yellow card for a grammar rule violation.
Shape up, bucko. This is not a job for the faint of heart or the poor at grammar. There are rules that must be followed. You're welcome. |
Since: Jan 13 932 |
Feb 3, 2013
We were 31 & 27 at the time. I had told my wife that I would like to see her with another man in her. She initiated out first MFM. It was thrilling from start to finish. The foreplay was great but when she laid on her back, spread her beautiful legs and asked him to get on I knew it was about to happen. She looked up at him and smiled, took his hard cock in her soft hand, rubbed it up and down her slit smearing the precum before guiding it inside her was a fantastic sight. This was the hottest and most exciting night of our lives. He was invited back the following weekend for more and didnt hesitate to accept the invitation. We had more with him and others for years. |
Since: Jan 13 136 |
Feb 3, 2013
I've often thought about this, fingering and what her reaction would be like...and mine!
Since: Jul 12 533 Perth, Australia |
Feb 4, 2013
have you told your wife about your thoughts Alex? |
"Love Flashing" Since: Sep 12 1,552 United States |
Feb 4, 2013
Any ladies out there who were apprehensive about having a threesome and who now glad they do it? I would love to hear any tips or advice as how to help guide my wife into taking another mans kooock. Yes pun intended.. I would take a hands on approach.. MMF wanted in Phoenix.
Belfast, UK |
Feb 4, 2013
my wife, over the years has had several boyfriends who were better endowed than myself. Her current friend of long standing has a thick 7 inches, and recently we all went on holiday together for a week. I have never before seen her with another man. One early evening she took him to bed and from the doorway I saw him ease his large cock into her, I was so excited that I had a little erection (nearly 4 inches) for the full 30 minutes he was giving her one. I have not told my wife that I watched her, because I think she likes to keep her sexual satisfaction private.
Since: Aug 12 450 Location hidden |
Feb 4, 2013
Would love to watch a guy or guys slam n open up my 26yr old wife!
Feb 4, 2013
Sure is she hot also first time.
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Behrman RE, Kliegman R, Jenson HB. Nelson Textbook of Pediatrics . 18th ed. Philadelphia, PA: Saunders; 2007.
DynaMed Editorial Team. Down syndrome. EBSCO DynaMed website. Available at: https://dynamed.ebscohost.com/about/about-us . Updated September 2, 2010. Accessed November 17, 2010.
Ferri F, ed. Ferri's Clinical Advisor 2010 . Philadelphia, PA: Mosby Elsevier; 2009.
Malone FD, Canick JA, Ball RH, et al. First- and second-trimester evaluation of risk (FASTER) research consortium. First-trimester or second-trimester screening, or both, for Down's syndrome. N Engl J Med . 2005;353:2001-2011.
National Down Syndrome Society. Associated conditions. National Down Syndrome Society website. Available at: http://www.ndss.org/Resources/Health-Care/Associated-Conditions . Accessed July 28, 2008.
National Institute of Child Health and Human Development. Down Syndrome. National Institute of Child Health and Human Development website. Available at: http://www.nichd.nih.gov/health/topics/down/Pages/default.aspx . Accessed July 28, 2008.
3/12/2010 DynaMed's Systematic Literature Surveillance https://dynamed.ebscohost.com/about/about-us : Carter M, McCaughey E, Annaz D, Hill CM. Sleep problems in a Down syndrome population. Arch Dis Child. 2009;94(4):308-310.
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Kathy, I was very interested in the reply you sent to Judy dated July 16. You mentioned that you quoted information in a document by Theophilus Dean Fesler. Do you have the entire document or do you know where it might be available? The small part I found in the Indiana Historical Society Library was only 3 pages long but they were numbered 59, 60, and 61. This was one of the few references I have ever found to his second wife, Amanda George. Although I have been looking for information about her for years, I keep running into a brick wall. If you want any information I have on the Fesler's I would be happy to share it. Any light you can shed will be most appreciated. Sheila
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Victor Ganata
The Future of Primary Care
-- the tools are there, they're just not very evenly distributed
March 27, 2012
Berci Mesko, MD
liked this
I wonder if people will call this rationing. On one hand, patients would love getting treatment without having to endure primary care physician's waiting rooms. On the other hand, patients want to see/talk to the doctor in person when they don't need to, and refuse to come in when they do. And I know we have to take risks and make big changes to mitigate the inevitable primary care shortage but I can't help but feel that malpractice lawyers would see such a paradigm and go "kaching!" -
Victor Ganata
If this were to happen, specialists would need to be more well-versed in primary care issues for sure, because there are a lot of things you can't figure out without seeing the patient, and if the specialist is going to be the first person to eyeball the patient, they better be able to pick up on things that are far outside their area of expertise. From what I understand, the way they train specialists in the UK is amenable to this (they all have general practice/primary care experience beyond just training.) The residency and fellowship system in the U.S. would have to change drastically to make this tenable. -
Victor Ganata


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This recipe
was part of
Food Fight: Top Chef Challenge
which ended Jun 8, 2009 12:00 PM.
See winners »
I developed this for the Top Chef recipe contest. It's yummy. We like it best served with crusty sourdough bread and corn on the cob.
[Recipe is for one packet. Prepare one packet per person.]
- 1Preheat oven to 500º with jelly-roll sheet pan inside
- 2In microwave, cook butter and garlic for one minute, then add white wine to mixture.
- 3In the center of a large sheet of parchment paper, place one layer of onions in approximately an 8" x 5" area.
- 4Place scallops and sea bass lengthwise on top of onions.
- 5Place ginger sticks next to fish then line each side with half of shellfish,
- 6Top with tomatoes, basil and pour garlic/butter/wine mix over all. Season with S & P.
- 7Close packets by folding parchment across center till tight, and then completely, by folding sides towards center. Place on preheated sheet pan and bake for 10 minutes.
Member recipes are not tested by the CHOW food team.


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Circle shows the fabric over the jojn to the CF tube. I also used paper on the tail...both sides to stiffen the 1/16 balso up. Also used baking powder and CA as filler in this joint.... -
Similar use on the stab but I didnt use the SFC paper on that as it seemed to add too much weight in this instance. Its much better on the foam structures I have made so far..


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RAR1 and HSP90 form a complex with Rac/Rop GTPase and function in innate-immune responses in rice.
ABSTRACT A rice (Oryza sativa) Rac/Rop GTPase, Os Rac1, is involved in innate immunity, but its molecular function is largely unknown. RAR1 (for required for Mla12 resistance) and HSP90 (a heat shock protein 90 kD) are important components of R gene-mediated disease resistance, and their function is conserved in several plant species. HSP90 has also recently been shown to be important in mammalian innate immunity. However, their functions at the molecular level are not well understood. In this study, we examined the functional relationships between Os Rac1, RAR1, and HSP90. Os RAR1-RNA interference (RNAi) rice plants had impaired basal resistance to a compatible race of the blast fungus Magnaporthe grisea and the virulent bacterial blight pathogen Xanthomonas oryzae. Constitutively active Os Rac1 complemented the loss of resistance, suggesting that Os Rac1 and RAR1 are functionally linked. Coimmunoprecipitation experiments with rice cell culture extracts indicate that Rac1 forms a complex with RAR1, HSP90, and HSP70 in vivo. Studies with Os RAR1-RNAi and treatment with geldanamycin, an HSP90-specific inhibitor, showed that RAR1 and HSP90 are essential for the Rac1-mediated enhancement of pathogen-associated molecular pattern-triggered immune responses in rice cell cultures. Furthermore, the function of HSP90, but not RAR1, may be essential for their association with the Rac1 complex. Os Rac1 also regulates RAR1 expression at both the mRNA and protein levels. Together, our results indicate that Rac1, RAR1, HSP90, and HSP70 form one or more protein complexes in rice cells and suggest that these proteins play important roles in innate immunity in rice.
- Suman Ghosh, Heather E Shinogle, Gaurav Garg, George A Vielhauer, Jeffrey M Holzbeierlein, Rick T Dobrowsky, Brian S J Blagg[Show abstract] [Hide abstract]
ABSTRACT: Human Hsp90 isoforms are molecular chaperones that are often up-regulated in malignances and represent a primary target for Hsp90 inhibitors undergoing clinical evaluation. Hsp90α is a stress-inducible isoform of Hsp90 that plays a significant role in apoptosis and metastasis. Though Hsp90α is secreted into the extracellular space under metastatic conditions, its role in cancer biology is poorly understood. We report that Hsp90α associates with the Aha1 co-chaperone and found this complex to localize in secretory vesicles and at the leading edge of migrating cells. Knockdown of Hsp90α resulted in a defect in cell migration. The functional role of Hsp90α/Aha1 was studied by treating the cells with various novobiocin-based Hsp90 C-terminal inhibitors. These inhibitors disrupted the Hsp90α/Aha1 complex, caused a cytoplasmic redistribution of Hsp90α and Aha1, and decreased cell migration. Structure-function studies determined that disruption of Hsp90α/Aha1 association and inhibition of cell migration correlated with the presence of a benzamide side chain, as an acetamide substituted analog was less effective. Our results show that disruption of Hsp90α/Aha1 interactions with novobiocin-based Hsp90 C-terminal inhibitors may limit the metastatic potential of tumors.ACS Chemical Biology 11/2014; · 5.36 Impact Factor - SourceAvailable from: Guan-Feng Wang[Show abstract] [Hide abstract]
ABSTRACT: RAR1 and SGT1 are important co-chaperones of Hsp90. We previously showed that TaHsp90.1 is required for wheat seedling growth, and that TaHsp90.2 and TaHsp90.3 are essential for resistance (R) gene mediated resistance to stripe rust fungus. Here, we report the characterization of TaRAR1 and TaSGT1 genes in bread wheat. TaRAR1 and TaSGT1 each had three homoeologs, which were located on wheat groups 2 and 3 chromosomes, respectively. Strong inhibition of seedling growth was observed after silencing TaSGT1 but not TaRAR1. In contrast, decreasing the expression of TaRAR1 or TaSGT1 could all compromise R gene mediated resistance to stripe rust fungus infection. Protein-protein interactions were found among TaRAR1, TaSGT1 and TaHsp90. The N-terminus of TaHsp90, the CHORD-I and CHORD-II domains of TaRAR1 and the CS domain of TaSGT1 may be instrumental for the interactions among the three proteins. Based on this work and our previous study on TaHsp90, we speculate that the TaSGT1-TaHsp90.1 interaction is important for maintaining bread wheat seedling growth. The TaRAR1-TaSGT1-TaHsp90.2 and TaRAR1-TaSGT1-TaHsp90.3 interactions are involved in controlling the resistance to stripe rust disease. The new information obtained here should aid further functional investigations of TaRAR1-TaSGT1-TaHsp90 complexes in regulating bread wheat growth and disease resistance.Plant molecular biology. 02/2015; - SourceAvailable from: Yang Yuheng[Show abstract] [Hide abstract]
ABSTRACT: Rice (Oryza sativa L.) is the only widely cultivated gramineous crops that cannot be infected by rust fungi. To decipher the molecular basis of rice nonhost resistance (NHR) to Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust, proteomic analysis was performed using the two-dimensional electrophoresis (2-DE) technique. The expressed proteins from rice leaves 24 and 48 h post inoculation with Pst and from mock-inoculated leaves were identified. Quantitative analysis revealed a total of 27 differentially expressed proteins in response to Pst inoculation. Most of these proteins fall into the category "response to stimulus" and are involved in basic resistance processes, such as glycerol-3-phosphate and hydrogen peroxide signaling. A homologue of wheat leaf rust resistance protein Lr10 was also identified, implicating multiple layers of plant defense are implicated in rice NHR to Pst. These results demonstrate an intrinsic relationship between host and nonhost resistance. Changes in abundance of these proteins, together with their putative functions reveal a comprehensive profile of rice NHR to Pst and provide new insights into plant immunity.International Journal of Molecular Sciences 12/2014; 15(12):21644-21659. · 2.34 Impact Factor
Page 1
RAR1 and HSP90 Form a Complex with Rac/Rop GTPase and
Function in Innate-Immune Responses in Rice
Nguyen Phuong Thao,aLetian Chen,aAyako Nakashima,aShin-ichiro Hara,aKenji Umemura,bAkira Takahashi,c,1
Ken Shirasu,c,2Tsutomu Kawasaki,aand Ko Shimamotoa,3
aLaboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Ikoma 630-0101, Japan
bAgricultural and Veterinary Research Lab, Meiji Seika Kaisha, Kohoku-ku, Yokohama 222-8567 Japan
cSainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, United Kingdom
A rice (Oryza sativa) Rac/Rop GTPase, Os Rac1, is involved in innate immunity, but its molecular function is largely unknown.
RAR1 (for required for Mla12 resistance) and HSP90 (a heat shock protein 90 kD) are important components of R gene–
mediated disease resistance, and their function is conserved in several plant species. HSP90 has also recently been shown
to be important in mammalian innate immunity. However, their functions at the molecular level are not well understood. In
this study, we examined the functional relationships between Os Rac1, RAR1, and HSP90. Os RAR1-RNA interference (RNAi)
rice plants had impaired basal resistance to a compatible race of the blast fungus Magnaporthe grisea and the virulent
bacterial blight pathogen Xanthomonas oryzae. Constitutively active Os Rac1 complemented the loss of resistance, sug-
gesting that Os Rac1 and RAR1 are functionally linked. Coimmunoprecipitation experiments with rice cell culture extracts
indicate that Rac1 forms a complex with RAR1, HSP90, and HSP70 in vivo. Studies with Os RAR1-RNAi and treatment with
geldanamycin, an HSP90-specific inhibitor, showed that RAR1 and HSP90 are essential for the Rac1-mediated enhancement
of pathogen-associated molecular pattern–triggered immune responses in rice cell cultures. Furthermore, the function of
HSP90, but not RAR1, may be essential for their association with the Rac1 complex. Os Rac1 also regulates RAR1 expression
at both the mRNA and protein levels. Together, our results indicate that Rac1, RAR1, HSP90, and HSP70 form one or more
protein complexes in rice cells and suggest that these proteins play important roles in innate immunity in rice.
Plants use two innate immune systems to respond to pathogen
infection, known as pathogen-associated molecular pattern
(PAMP)–triggered innate immunity and effector-triggered immu-
nity (Zipfel and Felix, 2005; Chisholm et al., 2006; Jones and
Dangl, 2006). PAMP-triggered innate immunity is induced by
recognition of PAMPs by transmembrane receptors and gives
early responses to pathogen infection. Effector-triggered immu-
nity involves resistance (R) proteins as receptors that specifically
recognize pathogen effectors, either directly or indirectly, and
usually induces hypersensitive cell death (HR). The major R
proteins are intracellular receptors and contain nucleotide bind-
ing site (NBS) and leucine-rich repeat (LRR) domains. Although
there has been extensive research on plant innate immunity, the
molecular mechanisms of these two immune responses are not
well understood.
RAR1 (for required for Mla12 resistance) is an important
component of R gene–mediated disease resistance (Shirasu
et al., 1999; Tornero et al., 2002; Muskett et al., 2002), which
contains two zinc binding finger motifs termed CHORD-I and
CHORD-II (Cys- and His-rich domains) (Shirasu et al., 1999). In
plants, RAR1 interacts directly with SGT1 (for suppressor of the
G2 allele of skp1) and HSP90 (Azevedo et al., 2002; Liu et al.,
2002, Takahashi et al. 2003). The CHORD-I domain of RAR1
interacts directly with the N terminus of HSP90, and SGT1 binds
the CHORD-II domain of RAR1. SGT1 is required for disease
resistance mediated by diverse R proteins (Austin et al., 2002;
required for the function of an SCF (for Skp1/Cullin/F-box pro-
tein) (Kitagawa et al., 1999; Matsuzawa and Reed, 2001). HSP90
is an abundant, highly conserved, ATP-dependent molecular
chaperone that is essential for eukaryotic cell viability (Picard,
2002). More recently, RAR1, SGT1, and HSP90 were shown to
play an important role in regulating the stability of R proteins that
contain the NBS-LRR domain (Bieri et al., 2004; Holt et al., 2005;
Azevedo et al., 2006).
HSP90 and SGT1 play a key role in the activation of mamma-
lian immune responses induced by the Nod-like receptor (NLR)
protein family, a group that contains an NBS-LRR domain (Hahn,
2005; da Silva Correia et al., 2007; Mayor et al., 2007). SGT1 and
HSP90 interact with NLR proteins to form an inflammasome
complex and are required for activation of the complex. There-
fore, regulatory networks for immune responses in plants and
mammals share at least some common components, and
1Current address: Department of Molecular Genetics, National Institute
of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
2Current address: RIKEN Plant Science Center, 1-7-22 Suehiro,
Tsurumi, Yokohama 230-0045, Japan.
3Address correspondence to firstname.lastname@example.com.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Ko Shimamoto
WOnline version contains Web-only data.
OAOpen Access articles can be viewed online without a subscription.
The Plant Cell, Vol. 19: 4035–4045, December 2007, www.plantcell.org ª 2007 American Society of Plant Biologists
Page 2
understanding the molecular mechanisms involved in protein
complexes containing R proteins or NLR proteins, SGT1, and
HSP90 is becoming crucial in the study of innate immunity in
higher eukaryotes.
Rac/Rop small GTPases are highly conserved in the plant
kingdom and constitute the sole group of Rho family small
GTPases in plants (Gu et al., 2004). There are seven Rac/Rop
thaliana (Guetal.,2004).OsRac1,which islocatedin theplasma
membrane, is involved in ROS production through an NADPH
(Kawasaki et al., 1999; Ono et al., 2001). Os Rac1 functions as a
positive regulator of NADPH oxidase activation in PAMP signal-
ing (Kawasaki et al., 1999) and at the same time suppresses
expression of a scavenger metallothionein (Wong et al., 2004) in
transient accumulation of reactive oxygen species (ROS). Ex-
pression of constitutively active (CA)-OsRac1 enhances resis-
Suharsono et al., 2002), whereas a dominant negative (DN)
version of Os Rac1 (DN-OsRac1) suppresses HR induced by
incompatible races of rice blast fungus, indicating that Os Rac1
is one of the key regulators of rice innate immunity (Ono et al.,
2001). Os Rac1 regulates the stability and activation of Os
MAPK6, a rice mitogen-activated protein kinase, by sphingolipid
elicitors (Lieberherr et al., 2005). Cinnamoyl-CoA reductase, a
key enzyme in lignin biosynthesis, is activated by Rac1 in rice
innate immunity (Kawasaki et al., 2006), and DN-OsRac1 inhibits
N-mediated resistance to tobacco mosaic virus infection in
tobacco (Nicotiana tabacum; Moeder et al., 2005). There are a
number of other studies aimed at understanding the molecular
mechanisms of rice innate immunity (Yang et al., 2004; Gu et al.,
2005; Wangetal.,2006; Takahashietal.,2007),but thedetails of
how these pathways are regulated remain unknown.
GTPase. We show that both RAR1 and HSP90 play a critical role
in rice innate immunity and that they apparently form a complex
with Rac GTPase. Furthermore, loss of HSP90 function impairs
immune responses as well as the formation of a protein complex
with Rac GTPase-RAR1-HSP90. These results indicate a critical
role for the functional protein complex containing Rac GTPase,
RAR1, and HSP90 in rice innate immunity.
Os RAR1 Is Involved in Basal Resistance against Rice Blast
and Bacterial Blight Infection in Rice
RAR1 is required for the function of multiple R genes, such as
Mla, RPM1, RPS2, RPS5, and N genes (Shirasu et al., 1999;
Tornero et al., 2002; Muskett et al., 2002; Austin et al., 2002;
Azevedo etal., 2002;Liu etal.,2002; Holt etal., 2005).Therefore,
we were interested in determining whether RAR1 is involved in
disease resistance in rice.
To investigate the function of RAR1 in rice disease resistance,
we knocked down RAR1 expression with RNA interference
(RNAi) (Figures 1A and 1B; see Supplemental Figure 1A online).
Generally, Os RAR1 expression was greatly decreased in Os
RAR1-RNAi lines (Figure 1; see Supplemental Figure 2 online).
Japonica rice var Kinmaze, which was used for the production of
transgenic plants, carries the Pi-a blast resistance gene that is
incompatible with race 031 but is compatible with race 007 of
Magnaporthe grisea. Two T1 transgenic lines carrying the Os
RAR1-RNAi construct (Figure 1B, lines 18 and 61) showed no
difference in response to infection with race 031 from the wild-
type plant or a T1 segregant without a transgene (Figure 1B, line
15), making it unlikely that Pi-a–mediated blast resistance re-
quires RAR1. To further test for possible Os RAR1 requirements
in R gene–mediated blast resistance, we performed the same
infection analysis using the Os RAR1-RNAi transformants in the
Kanto IL 5 and Kanto IL 14 backgrounds that carry Pi-z and Pi-b
blast resistance genes, respectively. These experiments clearly
showed that RAR1 is not required by either of these two R genes
for the three R genes for rice blast resistance is consistent with
previous results indicating that RAR1 is required for many but not
all R genes examined (Shirasu and Schulze-Lefert, 2003).
To determine whether RAR1 is involved in basal resistance to
virulent blast isolates, Os RAR1-RNAi plants were infected with
compatible race 007. Leaves of untransformed control plants
developed typical disease symptoms (Figures 1C and 1D, race
carried the Os RAR1-RNAi transgene had larger lesions (Figures
1C and 1D, lines 26 and 28), whereas the two segregants that
carried no transgenes had lesion lengths similar to the wild-type
control (Figures1Cand1D,lines 15and27).Resultsofinfections
assays with T0 transgenic plants are shown in Supplemental
Figures 1A to 1C online. Furthermore, infection of T1 generation
plants with the rice bacterial blight pathogen Xanthomonas
oryzae pv oryzae race 1 (T7174), which is also compatible with
var Kinmaze, gave results similar to blast infection (Figures 1E
and 1F). Results of infection assays with T0 transgenic plants are
shown in Supplemental Figures 1D and 1E online. These results
demonstrate that RAR1 is involved in basal resistance to both
rice blast fungus and bacterial blight. This is consistent with the
previous reports that RAR1 functions in the basal resistance of
Arabidopsis to Pseudomonas syringae pv tomato (Holt et al.,
2005) and of mlo barley (Hordeum vulgare) to M. grisea (Jarosch
et al., 2005).
Os Rac1 and RAR1 Are Functionally Linked in Basal
Resistance to Rice Blast
Our previous studies have shown that Rac1 is an important
component of disease resistance in rice (Kawasaki et al., 1999;
Ono et al., 2001; Suharsono et al., 2002). Therefore, to examine
whether Rac1 and RAR1 are functionally linked in rice disease
resistance, we generated transgenic rice carrying both CA-
OsRac1 and Os RAR1-RNAi by introducing the Os RAR1-RNAi
construct into CA-OsRac1 transgenic calli and regenerating
plantsfor infection experiments (Figure 2). As previouslyreported,
CA-OsRac1 rice plants were more resistant to a compatible race
of rice blast (Ono et al., 2001). Transgenic plants carrying both
the CA-OsRac1 and Os RAR1-RNAi constructs were more re-
sistant to rice blast than Os RAR1-RNAi plants and had the
4036 The Plant Cell
Page 3
Figure 1. RAR1 Is Involved in Basal Resistance against Rice Blast and Bacterial Blight Infection in Rice.
(A) Diagram of the Os RAR1-RNAi construct.
(B) Transgenic plants expressing Os RAR1-RNAi were inoculated with an incompatible race 031 of the rice blast fungus M. grisea. Lesions are shown on
leaf blades 12 d after inoculation. WT indicates untransformed wild type plant, and 18, 61, and 15 are T1 segregants derived from an Os RAR1-RNAi
plant. Response of leaf blades of the wild type Kinmaze with infection by the compatible race 007 was used as a control for this experiment. Total RNA
was extracted from leaves of the wild type Kinmaze and Os RAR1-RNAi plants. Os RAR1 mRNA was amplified by RT-PCR using specific primers. The
17S rRNA primers were included in the PCR reactions as an internal control.
(C) Response of transgenic plants expressing Os RAR1-RNAi to infection with the compatible race 007 of M. grisea. WT indicates untransformed control
plant, and 26, 28, 15, and 27 are T1 segregants derived from an Os RAR1-RNAi plant. Photographs were taken 12 d after infection. Presence or absence
of the Os RAR1-RNAi transgene are indicated by þ and ?, respectively. RNA analysis was performed as indicated in (B).
(D) Quantitative analysis of disease lesions induced by infection with a compatible race of rice blast fungus shown in (C). Leaves were inoculated with
compatible race 007 of M. grisea. Bars indicate SE obtained from 6 to 20 measurements.
(E) Response of transgenic plants expressing Os RAR1-RNAi to infection with X. oryzae pv oryzae race1 (T7174). WT indicates untransformed control
plants, and 26, 28, 15, and 27 are T1 segregants derived from an Os RAR1-RNAi plant. Photographs were taken 12 d after infection. Presence or
absence of the Os RAR1-RNAi transgene are indicated by þ and ?, respectively.
(F) Quantitative analysis of disease lesions induced by infection with a compatible race of bacterial blight shown in (E). Leaves were inoculated with the
X. oryzae race 1. Bars indicate SE obtained from four to nine measurements.
RAR1 and HSP90 in Rice Innate Immunity4037
Page 4
same level of resistance as untransformed control plants (Figure
2), indicating that RAR1 is required for Rac1-mediated basal
resistance to rice blast. Similar results were obtained for basal
resistance to bacterial blight (data not shown).
Attempts to generate Os SGT1-RNAi rice cell cultures and
whole plants by the same transformation protocol used for gen-
erating Os RAR1-RNAi cell culture and plants were unsuccessful.
This is likely due to the fact that Os SGT1 is an essential single-
copy gene in rice. Therefore, the function of SGT1 in rice innate
immunity has not as yet been determined by genetic methods.
Os Rac1 Forms a Complex with RAR1, HSP90, and
HSP70 in Vivo
Because Os Rac1 and Os RAR1 are functionally linked in basal
resistance, we examined whether RAR1 and Rac1 interact in
vivo. For an analysis of RAR1 interaction with Rac1 in vivo,
suspension cell cultures expressing myc-tagged CA-OsRac1,
DN-OsRac1, and CS-OsRac1 driven by the maize (Zea mays)
Ubiquitin promoter (Lieberherr et al., 2005) were used for
coimmunoprecipitation (co-IP) experiments. Results of co-IP
experiments using anti-myc and anti-RAR1 antibodies indicate
that CA-OsRac1 and DN-OsRac1 associate with endogenous
Os RAR1, but there is no association in extracts from CS-
OsRac1, which has impaired membrane localization, suggesting
that membrane localization of Rac1 isrequiredfor its association
experiments, we always detected two protein bands for Rac1 by
anti-myc antibody (Figure 3A, bottom panel; Lieberherr et al.,
2005). However, the reasons for this observation are not known.
To confirm the association of Os Rac1 and Os RAR1, we
immunoprecipitated RAR1 in protein extracts from three trans-
genic Rac1 cell cultures with anti-RAR1 antibody and then
Figure 2. Quantitative Analysis of Disease Lesions Induced by Infection
with a Compatible Race of M. grisea.
Leaves of the nontransgenic wild type, CA-OsRac1, Os RAR1-RNAi, and
double transgenic CA-OsRac1/Os RAR1-RNAi plants were used in three
independent experiments (mean 6 SE; n ¼ 12 to 61). Double transgenic
plants were made by introducing the Os RAR1-RNAi construct into the
CA-OsRac1 transgenic line. The double transgenic plants were more
resistant to race 007 of M. grisea than Os RAR1-RNAi plants and more
susceptible than CA-OsRac1 plants.
Figure 3. Os Rac1 Forms a Complex with RAR1, HSP90, and HSP70
in Vivo.
(A) Co-IP of OsRac1 and RAR1, HSP90, and HSP70. Total protein
extracts from Os Rac1 transgenic mutants were incubated with anti-myc
antibody and protein A Sepharose beads. Precipitates were washed,
collected by centrifugation, and separated by SDS-PAGE. Total ex-
tracted and immunoprecipitated samples from wild type cell culture were
used as a control. Immunoblot analyses were performed with anti-RAR1
antibody. Os RAR1 was detected in CA and DN-OsRac1 immune
complexes but not in C212S-OsRac1 (top panel). All three Os Rac1
mutants contained HSP90 (second panel). HSP70 was also detected in
all three Os Rac1 mutants. Os SGT1 was not detected in Os Rac1 protein
complexes (bottom panel).
(B) Immunoprecipitation with anti-RAR1 antibody and immunobloting
with Os Rac1 indicates the association of RAR1 and Rac1 in complexes
from CA- and DN-OsRac1 cells but not from CS-OsRac1 cells. Signals
were also detected from crude extracts of transgenic but not of the
nontransgenic wild-type cell culture (top panel). As a positive control,
RAR1 was detected in crude extracts as well as in precipitated
complexes from all cell cultures (middle panel).
(C) Confirmation of HSP90 association with Rac1 in vivo. Immunopre-
cipitation was performed with anti-HSP90 antibody and immunoblotted
with anti-Os Rac1 (top panel) and anti-HSP90 (bottom panel) antibodies.
4038The Plant Cell
Page 5
examined the precipitate with anti-myc antibody (Figure 3B). Os
extracts but not in the CS-OsRac1 mutant, whereas RAR1
protein was detected in co-IPs from three Rac1 mutants and
the untransformed control (Figure 3B, top two panels). These
results indicate that RAR1 and Rac1 are part of the same protein
complex in rice cell cultures.
Because RAR1 interacts with HSP 90 and we confirmed
interaction of rice RAR1 and HSP90 in yeast two-hybrid assays
(data not shown), we tested whether HSP90 is part of the Rac1
complex in vivo. The co-IP experiments showed that HSP90
coprecipitated withRac1 in extracts from the three myc-OsRac1
transgenic cell cultures but not from untransformed cultured
cells (Figure 3A, second panel). The association between Rac1
and HSP90 was confirmed by co-IP with anti-HSP90 antibody
followed by immunoblots with anti-myc antibody (Figure 3C).
These results demonstrate that Rac1 and HSP90 associate with
each other in vivo.
HSP70 is known to participate with HSP90 in almost all
cochaperone complexes studied in eukaryotes (Pratt and Toft,
2003). Therefore, we examined whether HSP70 also coprecipi-
tated with Rac1 by anti-myc antibody (Figure 3A, third panel).
Os Rac1 complex. These data suggest that Rac1 forms a
multiprotein complex containing RAR1, HSP90, and HSP70,
which is mainly localized at the plasma membrane, although
other possibilities remain to be studied.
SGT1 interacts with RAR1 and HSP90 (Shirasu and Schulze-
Lefert, 2003), and the SGT1-HSP90 association was recently
shown to be crucial in NLR-mediated immune responses in
mammals (da Silva Correia et al., 2007; Mayor et al., 2007).
However, repeated experiments clearly indicate that SGT1 does
not coimmunoprecipitate with Os Rac1 (Figure 3A), Os RAR1
(Figure 3B), or HSP90 (data not shown) under the conditions
used. Because two-hybrid assays with rice RAR1, SGT1, and
HSP90 showed that SGT1 is able to interact with RAR1 and
HSP90 (data not shown), it is possible that SGT1 interaction with
the Rac1 complex may be unstable and transient in rice cell
cultures. Alternatively, SGT1 may complex with RAR1 and
HSP90 in some other subcellular localizations.
RAR1 and HSP90 Are Essential for Rac1-Mediated
Enhancement of Sphingolipid-Triggered Immune
Responses in Rice Cell Cultures
Because RAR1 and HSP90 complex with Os Rac1, we investi-
gated whether PAMP-triggered immune responses, such as PR
gene expression and ROS production, require RAR1. For this
purpose, we analyzed PR gene expression in cultured cells in
response to sphingolipid elicitors (SEs). SEs were isolated from
the membranes of rice blast fungus and were shown to induce
the accumulation of phytoalexins, cell death, increased resis-
(Koga etal.,1998;Umemuraetal.,2000;Suharsono etal.,2002).
was induced in the wild type and was strongly enhanced in CA-
OsRac1 cells (Figure 4A). Os RAR1-RNAi did not affect induction
of PBZ1 or Chitinase1 expression. Interestingly, however, in the
double transgenic CA-OsRac1/Os RAR1-RNAi cell line, PR gene
expression was lower than in the CA-OsRac1 cell culture and
close to wild-type levels (Figure 4A), indicating that the enhance-
ment of PR gene expression by CA-OsRac1 was counteracted
by Os RAR1-RNAi. Thus, RAR1 is essential for Rac1-mediated
enhancement of PR gene expression in rice cell cultures,
suggesting a functional link between Rac1 and RAR1 in PR
gene induction.
SE induces H2O2production in rice cell cultures (Suharsono
et al., 2002; Wong et al., 2004; Lieberherr et al., 2005), raising the
possibility that RAR1 is involved. H2O2production was much
higher in the CA-OsRac1 cell culture (Figure 4B). However, this
Figure 4. RAR1 and HSP90 Are Essential for Enhancement of PAMP-
Triggered Immune Responses Mediated by Rac1 in Rice Cell Cultures.
(A) Suppression of SE-induced PBZ1 and Chitinase 1 by Os RAR1-RNAi
in CA-OsRac1 cell cultures. SE induction of PBZ1 and Chitinase 1 was
examined in the wild type, CA-OsRac1, Os RAR1-RNAi, and Os RAR1-
RNAi/CA-OsRac1 double mutants after 3, 6, or 9 h of treatment with SE.
Activation of SE-induced PBZ1 and Chitinase1 expression in the CA-
OsRac1 cell cultures (left) was suppressed by Os RAR1-RNAi (right).
(B) Suppression of SE-induced hydrogen peroxide production by Os
RAR1-RNAi in the CA-OsRac1 cell culture. Levels of H2O2were mea-
sured 3, 6, or 9 h after SE treatment.
(C) SE-induced PBZ1 and Chitinase 1 expression in rice cell cultures in
the presence or absence of GDA treatment. Cell cultures of wild type and
CA-OsRac1 were treated with GDA (right) or control DMSO (left) over-
night with a time course of SE treatment. Expression of PBZ1 and
Chitanase1 was strongly enhanced in CA-OsRac1 cells and reduced by
GDA treatment (right).
RAR1 and HSP90 in Rice Innate Immunity4039
Page 6
increase in H2O2 production was diminished in the double
transgenic CA-OsRac1/Os RAR1-RNAi cell cultures (Figure
4B), indicating that, like PR gene induction, RAR1 is involved in
Rac1-mediated H2O2production in rice cell cultures.
Because HSP90 is essential for disease resistance in plants
(Schulze-Lefert, 2004) and for some NLR-mediated immune
responses in mammals (da Silva Correia et al., 2007; Mayor
et al., 2007), its role in PAMP-triggered immune responses
mediated by Os Rac1 was determined using geldanamycin
(GDA), an HSP90-specific inhibitor. Wild type and CA-OsRac1
cell cultures were pretreated with GDA overnight and then
treated with SE for 3, 6, and 9 h. PBZ1 and Chitinase1 were
induced by SE in wild-type cells, but expression was much
greater in CA-OsRac1 cells. A high level of PR gene expression
was observed even at time point 0 (i.e., without SE treatment) in
CA-OsRac1 transgenic cells, peaking by 6 h after SE treatment.
Treatment of cell cultures with GDA resulted in a substantial
decrease in PBZ1 and Chitinase1 mRNA in CA-OsRac1 cell
culture (Figure 4C). These results indicate that HSP90 function is
essential for Rac1-mediated enhancement of PAMP signaling.
RAR1 and HSP90 are essential for Rac1-mediated enhancement
of PAMP-triggered immune responses in rice cell cultures.
HSP90 but Not RAR1 May Be Essential for Association with
the Rac1 Complex
The nucleotide-bound state of HSP90 is known to be essential
for function of its client adaptor proteins in the complex (Catlett
such as NOD1 and NALP3 and activation of immune responses
in mammals (da Silva Correia et al., 2007; Mayor et al., 2007).
and RAR1, rice cell cultures were treated with 10 mM GDA, and
theinteraction ofOsRac1withHSP90wasmeasuredwithco-IP.
There was no HSP90 signal with GDA treatment, indicating that
5A). GDA treatment also caused dissociation of RAR1 from the
Rac1 complex (Figure 5B). Furthermore, anti-RAR1 antibody
was used to test whether intact HSP90 is required for HSP90-
RAR1 associations. GDA treatment reduced precipitation with
anti-RAR1 to nearly undetectable levels, suggesting that HSP90
is essential for association HSP90 and RAR1 (Figure 5C). Finally,
we tested whether intact Os RAR1 is required for association of
HSP90 in the Rac1 complex using Os RAR1-RNAi/myc-CA-
OsRac1 cell culture and found that RAR1 is not required for the
association of HSP90 in the Rac1 complex (Figure 5D). Together,
these results indicate that HSP90 function is essential for associ-
Os RAR1 Expression Is Regulated by Os Rac1
Os RAR1 protein was apparently present in higher concentra-
tions in CA-OsRac1 cell cultures, suggesting that Os RAR1
expressionisaffected byRac1.Totestthishypothesis,OsRAR1
mRNA accumulation was measured by RT-PCR in wild-type and
CA-OsRac1 cell cultures. Os SGT1 mRNA was also measured
because SGT1 transcripts are induced by pathogen infection in
rice and Arabidopsis (Cooper et al., 2003; Zimmermann et al.,
2004). CA-OsRac1 overexpression cultures had much higher
Figure 5. HSP90 Function but Not Os RAR1 May Be Essential for Their Association with the Os Rac1 Complex.
(A) and (B) Wild-type and CA-OsRac1 cell cultures were treated with 10 mM GDA or DMSO overnight. Total protein extracts were incubated with anti-
myc antibody and protein A Sepharose beads. Precipitates were washed, collected by centrifugation, and separated by SDS-PAGE. Immunoblot
analyses were performed with anti-HSP90 (A) or anti-RAR1 (B) antibody to examine the interactions of Rac1 with HSP90 and RAR1. Signals were
detected only from immunocomplexes without GDA treatment. Immunoblots with anti-myc antibody were used as a control ([A], bottom panel).
(C) Immunoprecipitation performed with anti-RAR1 antibody after GDA treatment. Immunoblot analyses were performed with anti-HSP90 antibody.
HSP90 was not detected in RAR1-precipitated complex after GDA treatment.
(D) RAR1 is not required for association of HSP90 in the Rac1 complex. Total protein extracts from the wild type, CA-, DN-, or CS-OsRac1, Os RAR1-
RNAi, and the CA-OsRac1/Os RAR1-RNAi double transgenic mutant were incubated with anti-myc antibody. Precipitates were washed, collected by
centrifugation, and separated by SDS-PAGE. Total extracted and immunoprecipitated samples from wild-type cultured cells were used as control.
Immunoblot analyses were performed with anti-HSP90 antibody to examine Os Rac1-HSP90 association. Os RAR1-RNAi in the CA-OsRac1/Os RAR1-
RNAi double transgenic mutant had no effect on Rac1-HSP90 association (last column).
4040The Plant Cell
Page 7
mRNA levels of both Os RAR1 and Os SGT1 (Figure 6A),
suggesting that Os Rac1 regulates RAR1 and SGT1 expression.
To further examine the regulatory relationship between Os Rac1,
Os RAR1, and Os SGT1, a cell culture in which CA-OsRac1 can
be induced (ICA) by treatment with dexamethasone (DEX) (Wong
et al., 2004) and an Os Rac1-RNAi cell culture (Miki et al., 2005)
were examined. Induction of CA-OsRac1 with DEX treatment
clearly increased both transcript (Figure 6B) and protein (Figure
6B) accumulation levels of RAR1 compared with the ethanol-
treated control (ICA/DEX and ICA/ethanol), and RAR1 protein
mRNA accumulation correlated with Rac1 expression, but SGT1
protein levels did not change in these cell cultures (Figure 6B).
These results indicate that RAR1 could be transcriptionally
regulated by Rac1, but any control of SGT1 by Rac1 would be
at the posttranscriptional level. The presence of some RAR1
that there is another regulatory component besides Rac1 or that
Rac1 also has some posttranscriptional roles in RAR1 expres-
sion. Nevertheless, these results suggest a close regulatory link
between Rac1 and RAR1 at the transcriptional and possibly
posttranscriptional levels.
In barley, Arabidopsis, and tobacco, RAR1, together with HSP90
and SGT1, plays a key role in R gene–mediated resistance
(Shirasu and Schulze-Lefert, 2003). Our results demonstrate that
RAR1 is required for basal resistance against compatible races
of rice blast and bacterial blight (Figures 1D to 1F) and that this
resistance is mediated by Os Rac1 (Figure 2). Interestingly, Os
resistance. Recently, it was shown that Os RAR1 is not required
for Pish-mediated blastresistance (Takahashi etal.,2007).Since
RAR1 is not required for all R genes (Shirasu and Schulze-Lefert,
2003), these results in rice are not unexpected. We demon-
strated thatRAR1 iscritical forOsRac1–mediated enhancement
of PAMP signaling (Figures 4A and 4B). Therefore, RAR1 activity
in rice innate immunity may be on a broader scale than in those
other species.
Our results are consistent with the involvement of RAR1 in
basal resistance in Arabidopsis and mlo barley (Holt et al., 2005;
response to virulent pathogens, and its molecular mechanism is
weak R proteins that recognize cognate effectors (Jones and
Dangl, 2006). In this model, RAR1 function associated with basal
resistance is also mediated by R proteins. More recently, RAR1
was shown to be a target of the P. syringae effector AvrB (Shang
et al., 2006). Therefore, considering the observation that there
are many PAMP receptors in plant cells and that RAR1 could
potentially form complexes containing such receptors and other
key signaling proteins, the extent of RAR1 involvement in various
defense signaling pathways remains to be studied.
Our yeast two-hybrid analysis showed that Os RAR1 and
HSP90 do not directly interact with Os Rac1, although Os RAR1
interacts directly with HSP90 (data not shown). Thus, it seems
that Os Rac1 forms a complex with Os RAR1 and HSP90 by
indirect interactions. We recently obtained evidence that Os
Rac1 interacts with Sti1/Hop in vivo and in vitro (L. Chen and
K. Shimamoto, unpublished data). Sti1/Hop is a cochaperone in
the HSP90 complex with HSP90 and HSP70 and is highly con-
served in eukaryotes (Pratt and Toft, 2003; Zhang et al., 2003).
Thus, Os RAR1 and HSP90 are likely to form a complex with Os
Rac1 through Sti1/Hop.
Results of RT-PCR and immunoblotting showed that the
overexpression of Os Rac1 strongly increased Os RAR1 mRNA
and protein accumulation (Figure 6), suggesting that Rac1 may
function upstream of RAR1 in defense signaling and that the
presence of a regulatory link between Os Rac1 and Os RAR1 in
SE-induced signaling. Interestingly, treatment with GDA did not
alter mRNA or protein accumulation of either Os RAR1 or Os
SGT1 (data not shown). These results may suggest the presence
of multiple regulatory pathways in which various components
interact with each other at multiple levels of regulation.
HSP90 Is a Critical Regulator in Os Rac1–Mediated PAMP
Signaling in Rice
Heat shock proteins are well known for regulating the maturation
of protein complexes, for degrading damaged or misfolded
peptides, and for involvement in the activity of many signal
Figure 6. Os RAR1 Expression Is Regulated by Os Rac1.
(A) Correlation of mRNA expression levels of Os Rac1, Os RAR1, and Os SGT1. Overexpression of CA-OsRac1 strongly increased mRNA accumulation
of RAR1 and SGT1 in rice cell cultures. Levels of mRNAs were measured by RT-PCR.
(B) Levels of mRNA (RT-PCR) and protein accumulation (protein gel blot [WB]) for Rac1, RAR1, and SGT1. DEX-inducible CA-OsRac1 (ICA) and an
OsRac1-RNAi cell culture were examined by RT-PCR and immunoblotting after DEX induction and ethanol as a control.
RAR1 and HSP90 in Rice Innate Immunity 4041
Page 8
transduction proteins (Pratt and Toft, 2003; Rutherford, 2003).
Previous studies have demonstrated that HSP90 plays a general
role in R protein–mediated immunity in plants and that it acts
physically close to R proteins (Hubert et al., 2003; Kanzaki et al.,
2003; Lu et al., 2003; Takahashi et al., 2003; Liu et al., 2004).
However, no specific role for HSP90 in protein complex forma-
tion or specific immune responses in plants has been identified.
The close association of HSP90 with Os Rac1 (Figure 3)
prompted an investigation into the possible involvement of
HSP90 in Rac1-mediated enhancement of PAMP signaling in
rice cell cultures. To examine the function of HSP90, we used
GDA, which binds the N-terminal structural domain of HSP90
binding pocket, and it has little effect on prokaryotic pathogens
(Stebbins et al., 1997; Picard, 2002). GDA experiments on rice
cell cultures demonstrated the requirement of HSP90 for Rac1-
mediated enhancement of PAMP signaling to induce PR genes
(Figure 4C). These results could be explained by the dissociation
of RAR1 and HSP90 from the Rac1 complex caused by GDA
(Figure 5), since it has been demonstrated that GDA treatment
alters the conformation and dissociation of cochaperones, such
as p23, Hop, and HSP70, from the steroid receptor complex in
mammalian cells (Whitesell et al., 1994; Bagatell et al., 2001;
Waza et al., 2006). These findings are similar to those in recent
studies of innate immunity in mammals (da Silva Correia et al.,
2007; Mayor et al., 2007), which demonstrate that GDA causes
inactivation of NLR-containing protein complexes and that
HSP90 is required for stability of the signaling complex.
We demonstrated the critical role of HSP90 in Os Rac1–
mediated enhancement of PAMP signaling; thus, we tested
whether HSP90 is also required for SE-dependant ROS pro-
duction. We examined H2O2production after GDA treatment
followed by SE treatment. However, GDA failed to suppress
SE-induced H2O2production in CA-OsRac1 cell cultures (data
not shown). One possible interpretation of this result is that GDA
itself increases ROS generation (Dikalov et al., 2002).
Withregardtothe possiblefunctionofHSP70 ininnateimmune
responses in rice, we have no information at the moment. The
three groups (N.P. Thao and K. Shimamoto, unpublished data),
much like Arabidopsis, which also contains 14 genes for HSP70
(Sung et al., 2001). We attempted to knock down expression
of nine genes in group 1 and three genes in group 2 by RNAi
methods employing conserved coding sequences as targets
(Miki et al., 2005). However, we were not able to obtain trans-
was clearly decreased (N.P. Thao and K. Shimamoto, unpub-
lished data). A different approach would therefore be required to
determine the function of HSP70, if any, in rice innate immunity.
A Network of Proteins Involved in Innate Immunity in Rice
CA-OsRac1 activates ROS production and HR in rice and
confers resistance to rice blast and bacterial blight (Kawasaki
et al., 1999; Ono et al., 2001; Suharsono et al., 2002). However,
the molecular mechanisms of Os Rac1 immune response reg-
ulation are largely unknown. In this study, we present evidence
suggesting that Os Rac1 associates with well-studied compo-
nents of plant innate immunity, such as RAR1 and HSP90, and
HSP70. We have previously demonstrated that Os Rac1 also
a WD repeat–containing receptor of activated C kinase homolog
(RACK1/RWD), which is involved in hormone signaling and
development (Chen et al., 2006), specifically binds CA-OsRac1
and is essential for basal resistance to rice blast and PAMP-
triggered immunity in rice (A. Nakashima and K. Shimamoto,
unpublished data). RACK1/RWD is likely to be a second link
between Rac1 and RAR1 because RWD interacts directly with Os
(A. Nakashima and K. Shimamoto, unpublished data), thus con-
firming the observation that RAR1 is part of the Rac1 complex.
These results connect Rac/Rop GTPase with known players in
plant defense pathways and reveal the general importance of
overexpression of CA-OsRac1 increased transcript levels of Os
RAR1 and Os SGT1 and that CA-OsRac1 complements the loss
of Os RAR1 in basal resistance to rice blast (Figure 2) suggest
that Rac1 is a central mediator of disease resistance in rice and
that it coordinates the activity of other important factors, such as
RAR1, HSP90, and the MAPK cascade. Recently, wehave found
a direct interaction between CA-OsRac1 and the N terminus of
the NADPH oxidases Os RBOHs and St RBOHB (Wong et al.,
2007). Thus, it is possible that Os RAR1 and HSP90 are required
for full activation of the Os Rac1 complex, at least partially
through regulation of NADPH oxidase.
With respect to the intracellular localization of the Os Rac1
complex, the results obtained in this study and previous studies
(Ono et al., 2001) suggest that the primary location of Rac1
complex is likely to be at the plasma membrane. However, since
recent studies indicate that some R proteins also function in the
nucleus (Shen and Schulze-Lefert, 2007), whether the Rac1
complex could be also located in the nucleus or the cytoplasm
remains to be studied.
SGT1 plays diverse roles in plants, whereas RAR1 has much
more specialized resistance functions (Shirasu and Schulze-
Lefert, 2003). SGT1 was not detected in immunoprecipitated
RAR1 complexes in rice cell cultures under the conditions we
used, whereas yeast two-hybrid analysis showed that rice SGT1
and RAR1 directly interact (data not shown). One explanation for
this is that the interaction between Os SGT1 and Os RAR1 is
a single complex or whether they simultaneously coregulate
other complexes required for downstream signaling events. One
current model derived from these observations is that a network
of proteins including some known components of plant innate
immunity, such as Os Rac1, RAR1, SGT1, HSP90, HSP70, Os
MAPK6, and RBOH (NADPH oxidase), can form one or more
protein complexes.
Biological Materials
mutants Os Rac1-G19V (CA-OsRac1), Os Rac1-T24N (DN-OsRac1), and
4042The Plant Cell
Page 9
Os Rac1-C212S (CS-OsRac1) were described previously (Kawasaki
et al., 1999; Ono et al., 2001). Os Rac1 protein was tagged with the
myc epitope at the N terminus, and the expression of each construct was
under the control of maize (Zea mays) Ubiquitin promoters. To make an
Os RAR1-RNAi construct, a cDNA fragment amplified by PCR using
two primers, Os RAR1-F (59-TCTGAGTGAGCCTAGGGTTTG-39) and
Os RAR1-R (59-GACCGAAGTCTCCACACACA-39), was inserted into
pANDA developed as a vector for RNAi (Miki and Shimamoto, 2004).
Agrobacterium tumefaciens–mediated transformation of rice calli was
performed according to a published method (Hiei et al., 1994). Plants
regenerated from transformed calli were selected by hygromycin resis-
tance. Rice suspension cell cultures expressing Os RAR1-RNAi and
CA-OsRac1/Os RAR1-RNAi were also produced.
Infection of Rice Plants with Blast Fungus and Bacterial Blight
Growth conditions for Magnaporthe grisea and methods for leaf punch
infection have been described previously (Takahashi et al., 1999). For
bacterial blight infection, leaves were inoculated by the clipping method
(Ono et al., 2001) with the Japanese Xanthomonas oryzae pv oryzae race
1 (T7174), which is compatible with var Kinmaze. Disease lesions were
measured 12 d after inoculation.
Rice Cell Cultures and Elicitor Treatment
Rice cell cultures expressing CA- and DN-OsRac1 were generated as
described previously (Kawasaki et al., 1999; Ono et al., 2001; Suharsono
et al., 2002). For analysis of gene expression, rice cell cultures were
collected after treatment with 5 mg/mL of an SE prepared from rice blast
Gene Expression
Total RNA was extracted from cultured cells and seedlings using RNeasy
plant RNA extraction kit (TAKARA). One mg RNA was digested with
PCR reactions were performed with specific primer sets: Os Rac1
(59-AGATAGGGCCTATCTTGCTGATCATC-39 and 59-ACAAGCGCTTC-
CGCAAAAGT-39),Actin (59-ACAGGAGAGAGCAGGCAGAG-39
CATGAA-39 and 59-GTCGCACACCGCCACC-39), Chitinase1 (59-TCT-
TAACATCACTGCAACTCAG-39 and 59-CTGCGAGCTCTGGACAC-39),
Ubiquitin (59-CCAGGACAAGATGATCTGCC-39 and 59-AAGAAGCTGAA-
GCATCCAGC-39), Os SGT1 (59-ATGGATCCATATGGCAACCGCCGC-
CGCG-39 and 59-ATGCGGCCGCTTAGTACTCCCATTTCTTAAGC-39),
and Os RAR1 (59-TGCAAAACTGGAAAGCACAC-39 and 59-GGAACTG-
Protein Analysis
Rice suspension cell cultures expressing CA-OsRac1, DN-OsRac1, and
C212S-OsRac1 were ground in cold extraction buffer (137 mM NaCl, 8.1
mM Na2HPO4anhydrous, 1.47 mM Na2HPO4, pH 7.4, 10% sucrose, and
complete protein inhibitor tablets [Roche]). Cell debris was removed by
centrifugation at 12,000g for 25 min. Protein concentrations were deter-
mined using Bradford protein assay with BSA as the standard. Proteins
wereseparated in10 or12.5%SDSpolyacrylamidegelsandblotted onto
nitrocellulose membranes (Millipore) with a semidry electroblotting-Trans
blot SD cell (Bio-Rad).
For immunodetection, membranes were incubated for 1 h with primary
antibodies against the myc epitope (mouse monoclonal [Invitrogen] or
rabbit polyclonal [Santa Cruz Biotechnology]). Hv RAR1 (Takahashi et al.,
2003), Hv HSP90 (Takahashi et al., 2003), SGT1 (Takahashi et al., 2003),
and HSP70 (Stressgen Biotechnologies), followed by anti-mouse/rat/
rabbit/IgG were conjugated to horseradish peroxidase (Sigma-Aldrich).
Specific protein bands were visualized with the ECL chemiluminescent
Western blotting detection reagent (GE Healthcare) and Hyperfilm ECL
(GE Healthcare).
For immunoprecipitations, 1 g extract (in a 1-mL volume) was incu-
48C for 3 h. Supernatants were collected and combined with 20 mL anti-
myc antibody and rotated end-over-end at 48C for 1 h. Fifty microliters of
protein A or G were added, and the incubation was continued overnight.
Immunocomplexes were washed three times with 1 mL ice-cold washing
buffer (extraction buffer plus 150 mM NaCl and 0.5% Triton X-100),
and separated by SDS-PAGE as described.
Quantification of H2O2
QuantificationofSE-induced H2O2production was performed withmodi-
cultured cells were precultured in a fresh R2S medium at 308C for 16 h.
Cells were transferred to 2mL offresh medium containingSE (10 mg/mL).
Aliquots were collected following incubation and filtered through a 0.22-
mmfilter.Thefiltered aliquot (100mL)wasmixed with1mLxylenol orange
buffer [0.25 mM FeSO4, 0.25 mM (NH4)2SO4, 25 mM H2SO4, 10 mM
sorbitol, and 12.5 mM xylenol orange] and incubated for 2 h at room tem-
perature. Absorbance was measured in a spectrophotometer (Beckman)
at 650 nm, and H2O2levels were determined based on a standard curve
made from known concentrations of H2O2dissolved in the R2S medium.
ForGDA treatment, GDA was diluted froma 10mMstockin DMSOinto
fresh R2S medium in the preculture step (described above) to a concen-
tration of 10 mM. DMSO alone was added into the R2S medium as a
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: AB029508 (Os Rac1),
(Os Chitinase1).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Basal Resistance to Rice Blast and Bacterial
Blight Infection in Os RAR1 RNAi T0 Plants.
Supplemental Figure 2. Os RAR1 Transcript Levels Are Consistent
with Protein Levels in Os RAR1-RNAi Rice Cultured Cell Lines.
We thank Mika Nobuhara, Yuko Tamaki, and Masako Hamane for
technical assistance. This research was supported by Grants-in-Aid
from the Ministry of Agriculture, Forestry, and Fisheries of Japan (Rice
Genome Project IP4001) and the Japan Society for Promotion of
Science (13G0023) to K.S.
Received September 5, 2007; revised November 22, 2007; accepted
December 7, 2007; published December 21, 2007.
RAR1 and HSP90 in Rice Innate Immunity4043
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RAR1 and HSP90 in Rice Innate Immunity 4045


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Hospitals welcome vetoes; wait to see what comes next
WILLMAR -- The state budget bills approved by the Legislature before it adjourned Monday night could have been disastrous for the state's hospitals.
Now that Gov. Mark Dayton has vetoed the budget passed by the Republican-controlled Legislature, the state's health care system waits to see what will come next.
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"Do you believe in immortality? No, and one life is enough for me"
Albert Einstein
"Life is the childhood of our immortality."
Johann Wolfgang von Goethe
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Woody Allen
"One of the proofs of the immortality of the soul is that myriads have believed it - they also believed the world was flat"
Mark Twain
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Friedrich Nietzsche
"The first step to eternal life, is you have to die."
Chuck Palahniuk
"Each life makes its own immitation of immortality."
Stephen King