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Many eukaryotic genomes encode cis-natural antisense transcripts (cis-NATs). Sense and antisense transcripts may form double-stranded RNAs that are processed by the RNA interference machinery into small interfering RNAs (siRNAs). A few so-called nat-siRNAs have been reported in plants, mammals, Drosophila, and yeasts. However, many questions remain regarding the features and biogenesis of nat-siRNAs.
Through deep sequencing, we identified more than 17,000 unique siRNAs corresponding to cis-NATs from biotic and abiotic stress-challenged Arabidopsis thaliana and 56,000 from abiotic stress-treated rice. These siRNAs were enriched in the overlapping regions of NATs and exhibited either site-specific or distributed patterns, often with strand bias. Out of 1,439 and 767 cis-NAT pairs identified in Arabidopsis and rice, respectively, 84 and 119 could generate at least 10 siRNAs per million reads from the overlapping regions. Among them, 16 cis-NAT pairs from Arabidopsis and 34 from rice gave rise to nat-siRNAs exclusively in the overlap regions. Genetic analysis showed that the overlapping double-stranded RNAs could be processed by Dicer-like 1 (DCL1) and/or DCL3. The DCL3-dependent nat-siRNAs were also dependent on RNA-dependent RNA polymerase 2 (RDR2) and plant-specific RNA polymerase IV (PolIV), whereas only a fraction of DCL1-dependent nat-siRNAs was RDR- and PolIV-dependent. Furthermore, the levels of some nat-siRNAs were regulated by specific biotic or abiotic stress conditions in Arabidopsis and rice.
Our results suggest that nat-siRNAs display distinct distribution patterns and are generated by DCL1 and/or DCL3. Our analysis further supported the existence of nat-siRNAs in plants and advanced our understanding of their characteristics.
Gene regulation through small non-coding RNAs has been recognized as an important mechanism for almost all cellular processes in eukaryotes. Most small RNAs (sRNAs) are processed by RNase III-type ribonuclease Dicer-like (DCL) proteins, and incorporated into Argonaute (AGO) proteins to guide gene silencing at the transcriptional level through DNA or histone modification, and at the posttranscriptional level by mRNA cleavage and degradation or translational repression [1-5]. Based on their precursor structures and biogenesis, sRNAs can be categorized into microRNAs (miRNAs) and small-interfering RNAs (siRNAs). miRNAs are derived from hairpin-structured precursors and do not depend on RNA-dependent RNA polymerases (RDRs) or RNA amplification mechanisms, whereas endogenous siRNAs (endo-siRNA) are generated from long double-stranded RNAs (dsRNAs) as a result of antisense transcription or the activity of plant-specific RNA polymerases such as RNA polymerase (Pol)IV and RDRs [6-8].
Different classes of endogenous siRNAs have been described in Arabidopsis based on their distinct characteristics and biogenesis pathways [3,9]. The 24-nucleotide heterochromatic siRNAs (hc-siRNAs) or repeat-associated siRNAs (ra-siRNAs) are generated by the PolIV/RDR2/DCL3 pathway and function in RNA-directed DNA methylation and histone modifications [2,10-12]. Trans-acting siRNAs (ta-siRNAs) are 21 nucleotides in length and require DCL4, RDR6, and miRNA-mediated cleavage that triggers the phased processing [3]. Long siRNAs (lsiRNAs) are 30 to 40 nucleotides in length and are dependent on DCL1, HEN1, RDR6 and PolIV [13]. lsiRNAs most likely induce decapping and 5' to 3' mRNA degradation of their targets [13]. Several cis-natural antisense transcripts (cis-NATs) have been reported to also generate siRNAs [14-18]. These so-called nat-siRNAs are induced by abiotic and biotic stresses [16,17,19] or accumulate in specific developmental stages [14,15]. The biogenesis of salt- and bacterium-induced nat-siRNAs appeared to require DCL1 and/or DCL2, RDR6, and PolIV in Arabidopsis [16,17]. Moreover, the expression of ARIADNE14 is de-repressed in dcl1, hen1, hyl1, sde4, rdr2 and sgs3, suggesting that the nat-siRNAs generated from the ARIADNE14/KOKOPELLI overlapping pair is dependent on DCL1, HEN1, HYL1, RDR2, SGS3 and PolIV [14]. The overlapping regions of some cis-NATs in mouse and Drosophila also produce endo-siRNAs, which are processed by Dcr-2 and loaded into AGO2 [20-24]. nat-siRNAs have also been found in budding yeast and Schistosoma japonicum [25,26]. Despite these reports, there remains a need for conclusive evidence for the occurrence of nat-siRNAs in plants [27], because their features are not well understood and a general model for their generation is still not available.
Here we report the identification of 33,826 and 162,397 siRNAs corresponding to 17,141 and 56,209 unique sequencing reads that were derived from cis-NATs of biotic and abiotic stress-challenged Arabidopsis and abiotic stress-treated rice, respectively. These siRNAs, displaying either a distributed or a site-specific pattern, were enriched in the overlapping regions and often exhibited strand bias. Genetic analysis revealed that nat-siRNAs in Arabidopsis were mainly generated by DCL1 and/or DCL3, and a subgroup of them was RDR- and PolIV-dependent. Furthermore, accumulation of some nat-siRNAs was regulated by biotic and abiotic stress conditions, which may contribute to plant responses to environmental stresses.
We sequenced a total of 21 sRNA libraries prepared from biotic and abiotic stress-treated Arabidopsis and abiotic stress-challenged Oryza sativa plants. Biotic stress datasets for Arabidopsis included infection of non-pathogenic Pseudomonas syringae pv tomato (Pst) DC3000 hrcC-, a virulent strain Pst DC3000 carrying an empty vector, and an avirulent strain Pst DC3000 carrying an effector gene, avrRpt2, as previously described [28-30]. Abiotic stresses on Arabidopsis and rice included salt, cold and drought treatments (see Materials and methods). We obtained a total of more than 24.6, 23.2 and 30.9 million raw sequencing reads from these biotic and abiotic stress-treated Arabidopsis and abiotic stress-treated rice small-RNA libraries, respectively (Additional file 1). After trimming adaptor sequences and discarding reads shorter than 17 nucleotides or longer than 28 nucleotides or of low quality, we obtained 13,985,938, 14,664,923 and 17,446,592 reads from the biotic and abiotic stress-treated Arabidopsis libraries and the abiotic stress-treated rice libraries that perfectly match to the Arabidopsis and rice genomes or cDNAs, respectively (Additional file 1). Among all the small-RNA species profiled, 24-nucleotide sRNAs were the most abundant size class (Additional file 2a). The 5'-first nucleotides of sRNAs preferentially are adenosine or uracil (Additional file 2c). In addition to a large number of miRNAs [28,29], we discovered many new endogenous siRNAs belonging to different siRNA categories. In this study, we focused on siRNAs that correspond to cis-NATs.
Using the Arabidopsis genome annotation (TAIR/NCBI version 8.0) and the rice genome annotation (MSU RGAP 6.1), we searched for pairs of genes in Arabidopsis and rice that overlapped more than 25 nucleotides at the same genomic loci. We thus identified 1,439 and 767 pairs of cis-NATs in Arabidopsis and rice, respectively (Table (Table1).1). cis-NATs can be further categorized into three groups: convergent (3'-3' overlap), divergent (5'-5' overlap), and enclosed, in which one transcript is entirely encompassed by the other. Most cis-NATs (1,126 and 560 pairs in Arabidopsis and rice, respectively) are arranged in convergent orientation (Table (Table1).1). We excluded 65 and 10 cis-NAT pairs in which at least one gene encodes rRNA, tRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), miRNA, ta-siRNA, or transposons in Arabidopsis and rice, respectively, from subsequent analyses. These RNA genes or transposons are considered 'hot spots' of RNA degradation or small RNA generation and may mask the real distribution of nat-siRNAs. We also analyzed the NAT genes targeted by miRNAs to eliminate the possibility that the sRNAs that matched to the transcripts are secondary siRNAs triggered by 22-nucleotide miRNAs [31,32]. We found that At2g33810 in the At2g33810/At2g33815 pair was a target of miR156 and At1g53230 in the At1g53230/At1g53233 pair was a target of miR319. However, neither miR156 nor miR319 is 22 nucleotides, and the siRNAs generated from At2g33810 and At1g53230 were not arranged in a phasing pattern, which is a characteristic pattern of miRNA-triggered secondary siRNAs. Therefore, we retained At2g33810/At2g33815 and At1g53230/At1g53233 in our list. A total of 1,374 NAT pairs in Arabidopsis were used in this study. Among them, 186 genes (6.8% of 2,748 NAT genes) are annotated as 'other RNAs'. They are most likely non-coding RNAs or RNAs with potential to encode very short proteins or peptides.
We searched our small RNA deep-sequencing libraries for reads that matched perfectly (that is, no mismatches) to the cis-NATs. We found that 84 (6.1% of 1,374) and 119 (15.7% of 757) of the Arabidopsis and rice cis-NAT pairs, respectively, gave rise to at least 10 sRNAs per one million total genome-mapped reads from the overlapping regions of the cis-NATs in all the libraries (Table (Table1;1; Additional file 3). Interestingly, among the 84 Arabidopsis NAT pairs, 54 pairs have one transcript (32.1% of 168 genes) annotated as 'other RNAs' (Additional file 4), suggesting that non-coding antisense RNAs are more likely to generate siRNAs. A total of 33,826 and 162,397 siRNAs corresponding to 17,059 and 56,209 unique reads were derived from cis-NATs in Arabidopsis and rice, respectively (Table (Table2).2). As shown in Additional file 2, nat-siRNAs are mainly 21 and 24 nucleotides in length, whereas in the total small RNA population, the 24-nucleotide siRNAs are the most abundant species. The first-nucleotide distribution for the nat-siRNAs was similar to that of the total sRNAs (Additional file 2).
Within the three types of cis-NATs, enclosed cis-NATs have the highest percentages for producing siRNAs, that is, 39 (27.1% of 144) and 39 (36.4% of 107) of the enclosed cis-NATs in Arabidopsis and rice, respectively, generated siRNAs (Table (Table1).1). Among the 84 and 119 cis-NAT pairs that produce more than 10 siRNAs per million reads in the overlapping regions in Arabidopsis and rice, respectively, 16 and 34 pairs (19.0% and 28.6%) have siRNAs exclusively in the overlapping regions (Table (Table1,1, Figures 1a-i and and2a).2a). To determine if siRNAs were truly enriched in the overlapping regions of NATs, we took into consideration the length differences between the overlapping and non-overlapping regions of cis-NATs by comparing the densities of siRNAs in these two regions. A statistical test showed that the density of siRNAs in the overlapping regions was more than six times that in the non-overlapping regions, with P-values of 0.0077 and 0.0242 in Arabidopsis and rice, respectively (see Materials and methods). To further explore whether cis-NATs have a higher likelihood to give rise to siRNAs than other protein-coding genes, we computed the densities of sRNAs in the 3'-UTR overlapping regions of convergent cis-NATs and 5'-UTR overlapping regions of divergent cis-NATs, and the siRNA densities in the 3'-UTRs and 5'-UTRs of protein-coding genes that were not involved in NATs, respectively (see Materials and methods). We then compared the density of siRNAs in the convergent cis-NATs with the density of siRNAs in the 3'-UTR of non-NAT genes in Arabidopsis; similarly, we compared divergent cis-NATs with the 5'-UTR of non-NAT genes. The densities of siRNAs in divergent 5' and convergent 3' cis-NATs were statistically greater than that of the 5'- and 3'-UTRs of non-NAT genes, with P-values of 0.0366 and 0.0438, respectively. These results confirmed that cis-NATs were more likely than non-NAT genes to generate siRNAs. This is further supported by the study of Argonaute-associated sRNAs, which indicated that cis-NATs generated 2.29 times more sRNAs than other gene transcripts [33]. Our results were also consistent with that of animal endo-siRNAs, which are also enriched in the overlapping regions of cis-NATs, for example, more than ten-fold enrichment in Drosophila [21-23,25,34].
The distribution of siRNAs along the cis-NATs display two distinct patterns, one is the distributed pattern with siRNAs scattered along the overlapping regions or across the entire transcripts (Figures (Figures11 and 2a, b), and the other is the site-specific pattern with siRNAs derived from one or a few specific sites (Figures (Figures2c2c and and3)3) (see Materials and methods for additional details). We identified 9 site-specific patterns from Arabidopsis and 23 from rice (Additional files 5 and 6). Because RNA secondary structure analysis at these specific sites using RNAFold [35] revealed no obvious stem-loop structures, these regions were unlikely to harbor new miRNA genes or nat-miRNA genes as described [36], although we are aware of the limitation of RNAfold. Moreover, 46 and 63 (50.5% and 52.9%) cis-NAT pairs in Arabidopsis and rice, respectively, exhibited strand bias in generating nat-siRNAs with different directions (with more than two-fold change) (Table (Table11).
To experimentally characterize the nat-siRNAs, we examined the expression of 15 new nat-siRNAs identified in Arabidopsis with relatively high numbers of reads. In addition to nat-siRNAATGB2, another 6 out of the 15 nat-siRNAs that we examined showed clear siRNA signals by small RNA Northern blot analysis using locked nucleic acid (LNA) probes (indicated by black arrows in Figures Figures11 and and3;3; probes used are listed in Additional file 7), which enabled us to study their biogenesis by examining their expression in small RNA pathway mutants. The biogenesis of the site-specific nat-siRNA generated from the divergent pair At1g51400/At1g51402 (referred to as nat-siRNA1g51400; Figure Figure3c)3c) was dependent on DCL1 and HEN1, but did not require any RDRs or PolIV (Figure (Figure4a).4a). Similarly, the nat-siRNA generated from the convergent pair At2g41590/At2g41600 with a distributed pattern (referred to as nat-siRNA2g41590; Figure Figure1j)1j) was also DCL1-dependent but RDR- and PolIV-independent (Figure (Figure4b).4b). Both nat-siRNAs were 21 nucleotides in length. Previous studies have shown that the biogenesis of bacterial-induced nat-siRNAATGB2, and the accumulation of some of the salt-induced nat-siRNASRO5 and development-associated nat-siRNAKPL also depended on DCL1 [14,16,17].
We found that the convergent NAT pair At1g13330/At1g13340 (Figure (Figure1o)1o) could produce two classes of siRNAs, 21-nucleotide and 24-nucleotide siRNAs (referred to as nat-siRNA1g13340) from the same site (Figure (Figure5a).5a). Genetic analysis revealed that the 21-nucleotide siRNAs were DCL1-dependent, whereas the 24-nucleotide siRNAs were DCL3-dependent. Furthermore, we found that two other 24-nucleotide nat-siRNAs with a distributed pattern, nat-siRNA1g28100 generated from NAT pair At1g28100/At1g28110 (Figure (Figure1l)1l) and nat-siRNA1g17745 generated from NAT pair At1g17744/At1g17745 (Figure (Figure1n),1n), were also dependent on DCL3 (Figure 5b, c). Interestingly, all the DCL3-dependent 24-nucleotide nat-siRNAs examined are dependent on RDR2 and PolIV (Figure (Figure5),5), while only some of the 21-nucleotide nat-siRNAs are dependent on RDRs and PolIV [14,16,17]. Deep sequencing results showed that 82 out of the 84 cis-NAT pairs gave rise to two classes of siRNAs, 20- to 22-nucleotide and 23- to 28-nucleotide siRNAs in the overlapping regions, while the remaining two pairs gave rise to the 20- to 22-nucleotide class only (Additional file 4). Thus, our results suggested that the cis-NATs have the potential to be processed by both DCL1 and DCL3.
In both plants and animals, nat-siRNAs are derived from NAT mRNAs and mainly function post-transcriptionally [16,17,23]. This was also supported by our observation that some NATs with distributed nat-siRNAs predominantly appeared in exonic regions of protein coding genes, for example, At2g33810/At2g33815, At1g13330/At1g13340, OS08g06220/OS08g06230, and At4g35850/At4g35860 (Figures 1g, o, ,2b2b and Figure Figure3a).3a). However, based on the Arabidopsis genome annotation (TAIR version 8), we found that 383 of the 1,374 cis-NAT pairs (27.9%) contain introns in the overlapping regions. Among them, 45 (11.7%) have siRNAs mapped to introns. Examples are shown for At2g41590/At2g41600, AT434070/At4g34071 and At2g02795/At2g02800 (Figure 1j, k, m). In rice, 346 of 757 (45.7%) cis-NAT pairs contain introns in the overlapping regions; one example is shown for OS04g57140/OS04g57160 in Figure Figure2c.2c. A substantial number of nat-siRNAs originated from such introns within NATs, as listed in Table Table22 and Additional files 8 and 9. Similarly, it has been reported that a large number of siRNAs are also generated from the introns of several cis-NATs in Drosophila [23]. It is possible that the intronic nat-siRNAs are generated from pre-mRNAs in the nucleus before the introns are spliced out.
To confirm these intron-derived nat-siRNAs, we validated the candidates in small RNA biogenesis mutants. Although the total number of siRNAs from the entire overlapping regions might be large, the reads for individual siRNAs were still low. Most of the siRNAs examined were below the detection level of Northern blot analysis except one siRNA that is derived from an intron-exon junction of At4g34070 in the overlapping region of the divergent NAT pair At4g34070/At4g34071 (Figure (Figure1k).1k). We detected a distinct 30-nucleotide siRNA band using a 24-nucleotide probe. Because we used a 18- to 26-nucleotide size-fractionated small-RNA population for constructing our small-RNA libraries, lsiRNAs were excluded from our small-RNA sequencing dataset. To determine whether it was a new lsiRNA like the ones we detected before [13], or just a degradation product, we examined the dependence of this 30-nucleotide siRNA on components of the small RNA biogenesis pathways. We found that the generation of this siRNA depended on DCL1 and HEN1, but not on DCL3, or any RDRs, PolIV or PolV, indicating that it was a new lsiRNA but not a hc-siRNA (Figure (Figure4c).4c). It is worth noting that the reported AtlsiRNA-1 is also dependent on DCL1 and HEN1 [13]. These results suggested that a subgroup of nat-siRNAs was likely to be generated from pre-mRNAs.
We found that siRNAs from some cis-NATs accumulated to different levels in different libraries (Additional files 10 and 11). Eighty cis-NAT pairs in Arabidopsis and 37 cis-NAT pairs in rice displayed a more than two-fold difference of siRNA accumulation in the overlap regions under at least one stress condition as compared with mock-treated samples. This result suggested that these nat-siRNAs were likely to regulate expression of the NATs under stress conditions, although more experiments are needed to validate and confirm these changes.
To determine the regulatory function of nat-siRNAs on their NAT transcripts in cis, we performed computational analysis using dcl1-7 and dcl3-1 (inflorescence tissue) microarray data generated by the Carrington lab [37] (see Materials and methods), because DCL1 and DCL3 are responsible for nat-siRNA biogenesis. Of the 84 pairs of cis-NAT genes, 93 genes from 78 pairs were present on the 22-K microarray chip. Of the 93 cis-NAT genes, 22 (23.7%) exhibited a greater than 1.5-fold up-regulation in the dcl1-7 mutant compared to the wild type (WT) control (P-value < 0.05; Additional file 12). As a comparison, only 176 out of 2,215 (7.9%) total NAT genes were up-regulated. Although we cannot absolutely rule out the possible small RNA-independent function of DCL1 on the accumulation of miRNA targets [38], the result that more siRNA-associated NATs (23.5%) were regulated by DCL1 than total NATs (7.9%) strongly suggests that these small RNA-producing cis-NATs were more prone to down-regulation by DCL1-dependent nat-siRNAs. However, we did not find any of the 93 NAT genes that were up-regulated more than 1.5-fold in dcl3, suggesting that the DCL3-dependent nat-siRNAs may not be directly involved in the expression regulation of the NAT transcripts.
To confirm the results from the microarray analysis and to examine the expression of some siRNA-targeting NAT transcripts that were not present on the chip or were not identified from the analysis, we used real-time RT-PCR to examine their expression in the WT, dcl3-1 and dcl1-7 fwf2 double mutant plants. We chose to use the double mutant instead of the dcl1-7 single mutant because the fwf2 mutant, which carries a mutation in the ARF8 gene [39,40], largely rescued the pleiotropy and fertility defects of dcl1-7 [41,42]. We found that the accumulation of At1g51402 and At1g13330 increased six and four times more, respectively, in the dcl1-7 fwf2 mutant than in WT plants (Figure (Figure6),6), indicating that the antisense transcripts were no longer suppressed when nat-siRNA1g51400 and nat-siRNA1g13340 were not produced. For At2g41590/At2g41600 and At4g34070/At4g34071 pairs that produce siRNAs from both strands and have less strand bias, both sense and antisense transcripts were up-regulated in the dcl1-7 fwf2 mutant compared to the WT, suggesting that both transcripts were under the regulation of nat-siRNAs (Figure (Figure6).6). Although it has been shown that the arf8 mutation does not interfere with miRNA-mediated gene silencing or RNAi or small RNA generation [39,40], it is still possible that up-regulation of these NAT transcripts in the dcl1-7 fwf2 double mutant is due to the fwf2 mutation that is sRNA-independent. To test this possibility, we also examined the expression levels of these NAT genes in the fwf2 single mutant. As shown in Additional file 13, in the fwf2 single mutant, there was no clear induction of these NAT transcripts except for At1g13340. Both transcripts of the At1g13340/Ag1g13330 pair were induced in the dcl1-7/fwf2 double mutant and At1g13340 was induced to a similar extent in both single and double mutants (about two-fold), suggesting that the up-regulation of At1g13340 was mainly due to the arf8 mutation whereas induction of At1g13330 was attributable to the de-repression of small RNA-mediated silencing by the mutation in DCL1. sRNAs generated from this pair had a strong strand bias (Figure (Figure1o),1o), were predominantly generated from At1g13340 and targeted At1g13330. These results correlated well with our real-time RT-PCR results. In summary, these data indicated that the expression of NAT transcripts was regulated by the small RNAs generated from DCL1.
We found that only three out of the ten transcripts that we tested showed an increase in the dcl3-1 mutant compared to WT (Figure (Figure6),6), suggesting that some 24-nucleotide nat-siRNAs are still able to regulate the expression of NAT transcripts, although a majority of the NATs were not affected by the 24-nucleotide siRNAs.
To further analyze the function of these nat-siRNAs, we searched the publicly available deep sequencing datasets of the AGO-associated small-RNAs in Arabidopsis for the nat-siRNAs in the overlap regions we identified [43,44]. As shown in Additional file 14, the 20- to 22-nucleotide nat-siRNAs were mainly loaded into AGO2, the Argonaute protein that predominately binds 21-nucleotide sRNAs and contributes to anti-viral and anti-bacterial resistances [45-48]. Interestingly, the 23- to 26-nucleotide long nat-siRNAs were loaded into both AGO1 and AGO4, the Argonaute proteins responsible for mRNA cleavage and DNA methylation, respectively [49,50].
We identified 17,141 and 56,209 unique nat-siRNA sequences originating from cis-NATs in biotic and abiotic stressed Arabidopsis plants and abiotic stress-challenged rice, respectively. These siRNAs are enriched in the overlapping regions of cis-NAT pairs and display distributed or site-specific patterns.
Our biogenesis analysis suggested that the siRNAs from cis-NATs were dependent on DCL1 and/or DCL3. Deep sequencing results showed that 82 out of the 84 cis-NAT pairs that generated siRNAs in the overlapping regions gave rise to two classes of siRNAs, 20- to 22-nucleotide and 23- to 28-nucleotide nat-siRNAs, while only two pairs of NATs gave rise to one class of 20- to 22-nucleotide siRNAs (Additional file 4). The 20- to 22-nucleotide nat-siRNAs are generated by DCL1, whereas the 23- to 28-nucleotide nat-siRNAs were DCL3-dependent (Figures (Figures44 and and5).5). We also provide an example that a DCL1-dependent 21-nucleotide nat-siRNA and a DCL3-dependent 24-nucleotide nat-siRNA were generated from the same site in the overlapping region of a NAT pair (Figure (Figure5a).5a). These two classes of sRNAs have also been observed in transgene silencing, viral RNA silencing and endogenous inverted repeat (IR) loci, where long double-stranded precursors or intermediates are formed [51-55]. Different kinds of transgenic constructs can generate different types of sRNAs: sense transgenes mainly generate 21- and 22-nucleotide siRNAs by DCL2 [56]; IR transgenes generate both 21- and 24-nucleotide siRNAs by DCL4 and DCL3, respectively, or mainly 21-nucleotide siRNAs by DCL4 [55-58]; DCL1 and DCL2 are also involved in the generation of siRNAs induced by IR transgenes [56]. Similarly, the 21-nucleotide, 22-nucleotide and 24-nucleotide endogenous siRNAs are processed by DCL4, DCL2 and DCL3, respectively, from endogenous IR loci [52,53]. DCL1 is required for the accumulation of 21- and 24-nucleotide siRNAs processed from IR transgenes or 22- and 24-nucleotide siRNAs processed from certain endogenous IR loci [53,59]. Viral RNAs are processed into 21- and 24-nucleotide siRNAs by DCL4 and DCL3, respectively, and DCL2 processes viral RNAs into 22-nucleotide siRNAs when DCL4 is suppressed or inactivated [60-64]. Furthermore, some endogenous miRNAs in Arabidopsis and rice also generate two distinct sizes of DCL1-dependent short miRNAs and DCL3-dependent long siRNAs or miRNAs at the same sites [28,65]. Our results suggest that, like the endogenous IR loci, transgene loci and viral replicons as well as cis-NAT sense and antisense transcripts form double-stranded RNAs that can potentially be processed by DCL1 and/or DCL3 to generate 20- to 22-nucleotide short siRNAs and 23- to 28-nucleotide long siRNAs, respectively. In this regard, the previously reported apparent DCL2 dependence of a 24-nucleotide nat-siRNA [17] is intriguing and needs to be re-examined.
By examining the expression of NAT transcripts that are complementary to the siRNAs and analyzing published microarray data, we found that many 21-nucleotide nat-siRNAs were able to regulate the accumulation of their target transcripts (Figure (Figure6;6; Additional file 12), which was consistent with the function of reported 21-nucleotide nat-siRNAs processed by DCL1 [14,16,17]. However, only a small number of NATs targeted by siRNAs were up-regulated in the dcl3 mutant, suggesting that only a fraction of the 24-nucleotide class of siRNAs may regulate gene expression directly. Similar results were observed in the viral-derived 24-nucleotide siRNAs. Although 24-nucleotide viral siRNAs are as abundant as 21-nucleotide and/or 22-nucleotide siRNAs, they can neither silence viral RNAs nor suppress viral infection [60-64]. Endogenous 24-nucleotide siRNAs are mainly generated by the DCL3/RDR2/PolIV pathway, and are often associated with AGO4 and AGO6 and mainly direct DNA methylation or chromatin modification [49,50,54,66]. We found that the generation of the DCL3-dependent 23- to 28-nucleotide nat-siRNAs also required RDR2 and PolIV. Further studies are necessary to determine whether these 23- to 28-nucleotide nat-siRNAs can also direct DNA methylation or histone modification at their NAT loci.
We observed two major distribution patterns of nat-siRNAs - distributed and site-specific - in Arabidopsis and rice. We found that the site-specific nat-siRNAs tested were generated by DCL1. Because of its predominant role in processing hairpin structures into miRNAs, DCL1 is generally assumed to be capable of recognizing certain RNA secondary structures. The sense and antisense transcripts in a NAT pair are transcribed separately, and their overlapping regions may interact to form a dsRNA. In this partially dsRNA duplex, complex secondary structures may arise, which are recognized by the versatile DCL1 enzyme, and thus are processed to yield site-specific nat-siRNAs, or possibly distributed nat-siRNAs, depending on what secondary structures are formed and recognized by DCL1. It is also possible that these apparently site-specific nat-siRNAs may be generated across the whole overlapping region first as the distributed ones, but only specific siRNAs from particular sites are stable and thus accumulated, while the rest are degraded.
We showed that RDRs were not always required for nat-siRNA formation (Figure 4a-c), which suggested that the dsRNAs formed within the overlapping regions of the sense and antisense transcripts were sufficient for producing nat-siRNAs in such cases. As for the nat-siRNAs that do require RDRs [13,16,17], they were not completely eliminated in the rdr mutants. The partial dependence on RDRs suggested that these nat-siRNAs could be primarily generated from the dsRNA regions formed within overlapping sense-antisense transcripts, and then may be amplified by RDRs in a secondary amplification step. However, because the enrichment of cis-NAT-generated siRNAs compared to all genic siRNAs is relatively weak, one should not automatically assume that siRNA clusters corresponding to the cis-NAT genes are always caused by the cis-NAT configuration; it is possible that some siRNAs may coincidentally be in a cis-NAT region. Nevertheless, our observation of some siRNA clusters that appear exclusively in the overlapping regions of NATs strongly supports that at least some siRNAs are indeed caused by the cis-NAT configuration.
We also found that many nat-siRNAs were derived from introns or intron-exon junctions. It has been shown that the sense/antisense dsRNA pairs in vertebrates are processed in the nucleus but not the cytoplasm [67,68]. Therefore, some plant nat-siRNAs may also be produced in the nucleus before introns are spliced out from the NATs. Moreover, we found a new Arabidopsis lsiRNA, lsiRNA4g34070, which was generated from the exon-intron junction of a NAT pair. Similar to AtlsiRNA-1, lsiRNA4g34070 was processed by DCL1.
The extent of transcript overlap within each cis-NAT may differ from the TAIR annotations and requires careful examinations. For example, recent 3' rapid amplification of cDNA ends (RACE) experiments revealed that the major SRO5 transcripts (data not shown) are shorter than previously indicated [17], although the experiments also showed a longer SRO5 transcript that extends beyond the detected nat-siRNAs (GenBank accession JQ513374). Some genes such as AGO3 were annotated in the TAIR database as having no 3'-UTRs, but longer 3'-UTRs for some of these genes have been recovered by 3'-RACE in our labs and others. It appears that many genes have multiple forms of transcripts with various lengths of 3'-UTRs.
Plant nat-siRNAs have been shown to regulate gene expression in stress responses or under specific developmental stages [16,17]. In this study, we identified new nat-siRNAs that appeared to regulate the expression of their NAT mRNAs, which may contribute to gene expression reprogramming and fine-tuning in response to environmental stresses. NATs are widespread in eukaryotic organisms [19,69-72]. The expression of antisense transcripts may differ among different cell types or under different environmental conditions [19,73]. It is likely that different sets of nat-siRNAs may be generated from NATs in response to different developmental and environmental cues, and therefore play a broad regulatory role in gene expression under specific conditions. Several reported nat-siRNAs, such as nat-siRNASRO5, nat-siRNAATGB2 and nat-siRNAKPL, are induced under stress or appear at specific developmental stages and suppress the expression of their antisense transcripts post-transcriptionally [14,16-18,69]. Similar examples have also been observed in animal systems, for example, the siRNAs from the SIC34a1 gene pair are only generated from mouse kidney and testis [67]. Moreover, many cis-NAT gene pairs in mammalian brain, including the genes involved in Alzheimer's disease and LRRTM1/Ctnna2, a pair of cis-NAT genes that participates in schizophrenia, can generate nat-siRNAs [74]. A lot of these nat-siRNAs are up-regulated in olfactory discrimination training [74]. Therefore, most of the reported nat-siRNAs are generated under specific developmental or environmental conditions. In this study, we detected bacterial-induced nat-siRNAATGB2 [16], as well as another siRNA that is in phase with nat-siRNAATGB2 (Figure (Figure3a;3a; Additional file 10) by deep sequencing, whereas the developmental stage-specific nat-siRNA nat-siRNAKPL was not present in our database [14]. The salt-induced nat-siRNAs from the SRO5-P5CDH pair were also present in this dataset, but the number of reads was below our cutoff threshold (minimum ten reads per million total match reads in the overlapping region), so it was not included in our list. In order to be consistent with other abiotic stress treatment, the salt treatment condition in this study was different from that in the published work [17]. However, more SRO5-P5CDH siRNAs were detected from two other salt-treated small RNA libraries in a separate study (Additional file 15; Gene Expression Omnibus (GEO) accession number GSE33642). It is worth noting that our genome-wide analysis on nat-siRNA-targeting NAT transcripts used the microarray result generated from healthy inflorescences (GEO accession number GSE2473), which may miss some NAT transcripts that are regulated by siRNAs at a different developmental stage or under specific environmental conditions. In addition, we cannot rule out the possibility that some of the nat-siRNAs regulate the expression of their targets by translational inhibition and therefore cannot be directly detected at the mRNA level by microarray or real-time RT-PCR experiments.
Our studies of plant sRNAs showed that those derived from plant cis-NATs were enriched in their overlap regions and cis-NATs are more likely to generate sRNAs than other transcripts. Taken together with previously published data on plants, Drosophila and yeast, our results strongly support the existence of nat-siRNAs in eukaryotic organisms. Our genetic analysis indicated that cis-NATs were processed by DCL1 and/or DCL3. By analyzing a large number of small RNA libraries from stress-challenged plants, we found that some nat-siRNAs were likely to respond to different environmental stresses, and therefore contributed to plant stress resistance, development or other cellular processes by regulating corresponding NAT mRNAs. nat-siRNA-mediated gene regulation is one of the regulatory mechanisms for at least a subgroup of cis-NATs.
Arabidopsis thaliana mutants (dcl1-9 fwf2, dcl1-7 fwf2, dcl2-1, dcl3-1, dcl4-2, hen1, rdr2-1, rdr1-1, rdr6-15, PolIV-3 and PolV-11, fwf2) and their corresponding WT ecotypes were used in this study. Bacterial infection assays were performed on 4-week-old Arabidopsis grown at 23°C with 12 h light as described previously [28-30]. Abiotic stress assays were performed on 4-week-old Arabidopsis gl1 plants expressing Luciferase under the control of RD29 promoter grown at 23°C and 16 h light, and 3-month-old rice plants (Oryza sativa cv. japonica) grown at 28°C with 13 h light.
Bacteria infiltration was carried out on the leaves of 4-week-old Arabidopsis WT and mutant plants as described previously [46]. Briefly, leaves of 4-week-old Arabidopsis plants growing at 23°C with 12 h light were infiltrated with mock (10 mM MgCl2), non-pathogenic strain Pst DC3000 hrcC-, Pst DC3000 (empty vector) and Pst DC3000 (avrRpt2) at a concentration of 2 × 107 cfu/ml. The leaves were collected at 6 and 14 hours post-inoculation.
For abiotic stress treatment in Arabidopsis, plants grew at 23°C with 16 h light for 19 days, and were then divided into four groups. One of the groups was water-deprived (drought treatment) while the others were maintained with regular irrigation. At day 29, one group of plants was treated with salt solution (200 mM NaCl as irrigation solution for 24 h). Another group of plants was transferred to a growth chamber at 5°C and 16 h light for 24 h for cold treatment. The fourth group of plants was always maintained under regular conditions as a normal control. At day 30, all four groups of plants were collected. For small RNA library construction, the whole shoots of plants (stems, leaves and inflorescences) were used.
Three-month-old rice growing at 28°C and approximately 13 h of natural light were treated with drought (water withholding for 3 days), cold (5°C for 24 h) and salt (400 mM NaCl for 24 h, added as irrigation solution). The inflorescences of these plants were collected and used for sRNA library construction.
sRNA extraction and library construction were carried out as described previously [75,76]. Briefly, total RNA was isolated from infiltrated leaves and fractionated on 15% denaturing polyacrylamide gel. RNA molecules ranging from 18 to 26 nucleotides were excised and ligated to 5'- and 3'-RNA adaptors using T4 RNA ligase followed by RT-PCR and gel purification as described in the instructions from Illumina Inc. The small RNA libraries were sequenced by Illumina Inc. and UCR core facility.
We used LNA probes for small RNA Northern blot analysis to detect nat-siRNAs. A total of 100 μg total RNA was separated on a 17% acrylimide gel. The blots were probed at 39°C with PerfectHyb Plus Hybridization Buffer (Sigma-Aldrich, Saint Louis, MO, USA). The sequences of the oligos were listed in Additional file 7.
For QRT-PCR analysis, 5 μg total DNaseI-treated (Invitrogen, Grand Island, NY, USA) RNA was used for synthesizing cDNA using Oligo dT and SuperScript II (Invitrogen). Amplification of small RNA targets was carried out using a real-time PCR machine (MyiQ, Bio-Rad, Hercules, CA, USA). The sequences of primers used here are listed in Additional file 7.
Raw sequence reads were parsed to remove 3'-adaptors using an in-house program. Unique sequences from Arabidopsis small-RNA libraries were mapped to the Arabidopsis chromosome sequences [77]. To produce candidate datasets of sRNAs, we removed sequencing reads that matched to rRNAs, tRNAs, snRNAs, snoRNAs, transposons, retrotransposons, or repeats. rRNA, snRNA and snoRNA sequences were obtained from TAIR8 genome annotation, and tRNA sequences were downloaded from the Arabidopsis tRNA database [78] and TAIR [77]. Transposons, retrotransposons, and repeat sequences were downloaded from the TIGR v2 Arabidopsis Repeat Database [79] and RepBase [80].
The rice genome and annotation were retrieved from the MSU Rice Genome Annotation Project V6.1 (RGAP 6.1). Non-coding RNA (tRNA, rRNA, snoRNA and snRNA) were collected from fRNAdb [81] and the UCSC tRNA database. Transposons, retrotransposons and repeat sequences were based on transposable elements of MSU RGAP 6.1 and RepBase [80].
The enrichment of sRNAs in the overlapping regions of NATs was examined by the method outlined in [69]. The sRNAs in the combined libraries for Arabidopsis or rice were used here. Briefly, for an l-nucleotide genic or genomic region that gives rise to n sRNAs, we defined n/l as the density of small RNA loci in this region. For each pair of cis-NATs, we counted the number of sRNAs mapping to the overlapping region, No, and the total number of sRNAs matching the non-overlapping regions of the two genes, Ng, and measured the length of the overlapping region, Lo, and the total length of non-overlapping regions of the two genes, Lg. Then we computed the density of small-RNA loci in the overlapping region, No/Lo, and that of the non-overlapping regions of the two NAT genes, Ng/Lg. The one-tail paired two-sample t-test was applied to the two-paired samples of siRNA density, No/Lo and Ng/Lg. The average densities in the overlapping regions (Ao) and in the non-overlapping regions of the NAT genes (Ag) that spawn sRNAs were computed. The ratio Ao/Ag was considered as the enrichment score.
In order to explore whether cis-NATs were more likely to give rise to siRNAs than other protein-coding genes, we calculated the average densities in the overlapping regions (Ao) of cis-NATs. We then calculated the average densities Au of a set of randomly chosen regions from the other protein-coding genes, which have the same size as the cis-NATs. Statistical significance was measured by a P-value computed as the frequency of the occurrences that Au was greater than Ao. We applied this method to compare the siRNA density of convergent cis-NATs with that of 3'-UTRs and the siRNA density of divergent cis-NATs with that of 5'-UTRs.
To analyze the regulation of nat-siRNAs under a specific condition, the sRNA reads in a single library that matched 100% to the genome were normalized to one million. Then the normalized sRNA abundances were compared between different libraries. Furthermore, we calculated the maximum fold change among all treated samples compared to corresponding control conditions as a supplemental value (Additional files 10 and 11).
To classify the cis-NAT pairs into distributed and site-specific patterns, we first clustered sequencing reads mapped to regions of cis-NATs. Starting from the first reads closest to the 5' end of a cis-NAT, reads were clustered if the positions of their first nucleotides were within a ten-nucleotide long segment. Clusters with more than five reads were retained for further analysis. We calculated two metrics for each cis-NAT: 1) the number of small-RNA clusters and 2) the percentage of the number of reads within these clusters relative to the total number of reads mapped to the whole cis-NAT. We then categorized a cis-NAT to have a site-specific pattern if 1) it had no more than ten siRNA clusters and 2) the percentage of the reads within the clusters was greater than 50%; otherwise we categorized the cis-NAT to have a distributed pattern. The criterion of 10 siRNA clusters was chosen based on the observation that the frequency of clusters of 84 Arabidopsis cis-NATs has a clear separation between 10 and above (Additional file 5a). The percentage criterion (more than 50% sequence reads) was determined based on a visual exanimation of the relationship between the number of clusters and the frequencies of reads within the clusters in the 84 cis-NATS, as plotted Additional file 5b. As shown in the figure, this relationship seemed to follow two distributions, one is a linear correlation represented by the line in Additional file 5b and the other is a group with no more than ten clusters that retain more than 50% of the total sequence reads. The second distribution thus formed site-specific patterns of siRNAs. We thus used these two criteria because a site-specific pattern should have relatively more reads appearing in a relatively smaller number of clusters than a distributed pattern.
The processed microarray data of dcl1-7 and Ler WT Arabidopsis were downloaded from GEO accession number GSE2473 (samples GSM47014, GSM47015, GSM47016, GSM47017, GSM47018, GSM47019, GSM47023, GSM47024, GSM47025, GSM47026 and GSM47027). We identified differentially expressed genes using Rank Product [82]. The rationale behind Rank Product is that it is unlikely for a gene to be ranked in the top position in all replicates if the gene was not differentially expressed. It has been shown that Rank Product is less sensitive to noise and has a better performance than other methods when sample size is small [83]. We selected differentially expressed genes with a false discovery rate no greater than 0.05 for 1,000 permutations. Differentially expressed probe sets were then mapped to corresponding genes according to the annotation of the Affymatrix ATH1-121501 chip.
The processed AGO data for Arabidopsis were downloaded from the GEO databases under accession numbers GSE10036 and GSE12037. The number of AGO-associated nat-siRNAs was obtained by perfectly mapping the identified nat-siRNAs to each of the AGO pull-down datasets separately. The number was then normalized by the total number of AGO reads in an AGO dataset that can be perfectly mapped to the genome raised to per million for normalization. The enrichment of nat-siRNAs in an AGO pull-down relative to the other AGOs was calculated as the percentage of the normalized nat-siRNAs in the AGO of the total normalized nat-siRNAs in all AGO pull-downs.
The nat-siRNA regulated cis-NATs were identified from the set of differentially expressed genes from the microarray data of the dcl1-7 and Ler WT control Arabidopsis plants. The analysis was carried out based on two criteria: 1) more than 1.5-fold up-regulation with a P-value less than 0.05 in the dcl1-7 mutant and 2) presence of nat-siRNAs produced by the cis-NATs on the strands opposite to the NAT transcripts. The rationale behind these criteria is that the main function of DCL1-generated nat-siRNAs is to target cis-NATs on the opposite strands to down-regulate their expression. Thus, in a DCL1 mutant where no such nat-siRNAs are produced, the expressions of the targeted NATs are expected to be up-regulated.
AGO: Argonaute; cis-NAT: cis-natural antisense transcript; DCL: Dicer-like; dsRNA: double-stranded RNA; endo-siRNA: endogenous siRNA; GEO: Gene Expression Omnibus; hc-siRNA: heterochromatic siRNA; IR: inverted repeat; LNA: locked nucleic acid; lsiRNA: long siRNA; miRNA: microRNA; PCR: polymerase chain reaction; Pol: RNA polymerase; Pst: Pseudomonas Syringae pv tomato; RACE: rapid amplification of cDNA ends; RDR: RNA-dependent RNA polymerase; siRNA: small interfering RNA; snRNA: small nuclear RNA; snoRNA: small nucleolar RNA; sRNA: small RNA; ta-siRNA: trans-acting siRNA; UTR: untranslated region; WT: wild type.
HJ initiated the project; HJ, WZ and JZ designed the experiments; XiaomingZ and BB carried out the experiments; JX, XuefengZ and CZ performed the computational analyses; SG, LL, DN, HZ, CL and TW participated in the experimental analyses. HJ, WZ, JZ, RL, YiL, WL and JG coordinated the research. HJ, XiaomingZ, YL, JX, JZ and WZ wrote the paper. All authors have read and approved the manuscript for publication.
Distributions of the sequencing reads from small RNA libraries of abiotic and biotic treated Arabidopsis and abiotic challenged rice. Shown in the table are the total number of raw sequencing reads (total), the number of qualified reads that can map perfectly to the corresponding Arabidopsis or rice genome (mapped), intergenic regions (intergenic), intronic sequences (introns), transposons or repeats (repeats/mobile), tRNA, rRNA, snoRNA and snRNA sequences (ncRNA), trans-acting siRNA (ta-siRNA), microRNA sequences (miRNAs), intron-exon junctions (intron-exon junction) and exon-exon junctions (exon-exon junction). No mismatches were allowed in the mapping. The second number for each condition (column) is the percentage of reads relative to the total mapped reads.
(a-d) Distributions of the lengths (a, b) and the first nucleotides (c, d) of total siRNAs and nat-siRNAs in stress-challenged Arabidopsis and rice. (a) Length distributions of unique sequencing reads in Arabidopsis. The blue and red bars represent total siRNAs and nat-siRNAs, respectively. (b) Length distributions of unique sequencing reads in rice. The blue and red bars represent total siRNAs and nat-siRNAs, respectively. (c) First-nucleotide distribution of unique sequencing reads in Arabidopsis. (d) First-nucleotide distribution of unique sequencing reads in rice.
Normalized reads of siRNAs mapped to the overlapping regions of Arabidopsis and rice cis-NAT pairs, respectively (a) siRNAs mapped to the overlapping regions of 84 Arabidopsis cis-NAT pairs. (b) siRNAs mapped to the overlapping regions of 119 rice cis-NAT pairs. Each small RNA library was normalized to one million reads with 100% matching to the genome. The sum of normalized reads from each library was listed for each cis-NAT pair.
Arabidopsis cis-NATs with two classes of small RNAs mapped to the overlapping regions. 'gene1' and 'gene2' represent the first and second transcript in each cis-NAT pair. Listed are 84 Arabidopsis cis-NATs with siRNAs mapped to the overlapping region. Raw reads of 20- to 22-nucleotide and 23- to 28-nucleotide classes of siRNAs matching 100% to the genome were analyzed. The percentage of 20- to 22-nucleotide and 23- to 28-nucleotide siRNAs are presented.
(a) Distributions of the number of small RNA clusters in 84 Arabidopsis cis-NATs. The red line represents the separation between site-specific (left) and distributed (right) patterns. (b) The plot of two metrics of 84 cis-NATs in Arabidopsis. Each dot represents the number of clusters within the cis-NAT whole region and the percentage of small RNA reads in all clusters. The red dots in the rectangle were classified as site-specific patterns, whereas the blue dots were distributed patterns, represented by a linear correlated line.
Classification of cis-NAT pairs. We analyzed 84 Arabidopsis and 119 rice cis-NAT pairs that have more than 10 raw siRNAs mapped to the overlap region. The reads analyzed here are the raw sequencing reads mapped to the whole region from the combination of all libraries. (a) Arabidopsis cis-NAT pairs display different distribution patterns. (b) Rice cis-NAT pairs display different distribution patterns.
Oligos used in this study. A plus sign ('+') before the nucleotide depicts LNA residues.
Small RNAs mapped to the introns or intron-exon junction regions of Arabidopsis cis-NATs. Copy number indicates the number of raw reads in the combination of all libraries. (a) siRNAs mapping to introns in the overlapping region of cis-NATs in Arabidopsis. (b) siRNAs mapping to introns in the whole region of cis-NATs in Arabidopsis.
Small RNAs mapped to the introns or intron-exon junction regions of rice cis-NATs. Raw reads from all the libraries were analyzed. (a) siRNAs mapping to introns in the overlapping region of cis-NATs in rice. (b) siRNAs mapping to introns in the whole region of cis-NATs in rice
Normalized reads of siRNAs mapped to 84 Arabidopsis NATs under different conditions. Each library was normalized to a million reads that perfectly mapped to the genome. The fold change with maximum absolute value between treated sample and corresponding control is listed as a supplemental value. The pairs with more than two-fold change were annotated in red (up) or in blue (down). (a) Reads of small RNAs mapped to overlapping region of NATs under different conditions. (b) Reads of small RNAs mapped to whole region of NAT under different conditions.
Normalized reads of siRNAs mapped to 119 rice NATs under different conditions. siRNAs matching 100% to the genome were analyzed and each library was normalized to a million reads before analyzing. The fold change with maximum absolute value between treated and untreated libraries is listed. The pairs with more than a two-fold change are displayed in red (up, +) or blue (down, -). (a) Reads of small RNAs mapped to overlapping region of NATs under different conditions. (b) Reads of small RNAs mapped to whole region of NATs under different conditions.
Arabidopsis cis-NATs that are up-regulated in the dcl1-7 mutant. (a) List of probes of the 93 genes of the 84 cis-NAT pairs. Annotations of the Affymetrix ATH1-121501 chip were used and no probes were found for the rest of the cis-NAT genes. (b) The 23 cis-NAT genes up-regulated in dcl1-7.
Expression analysis of NAT transcripts in the fwf2 single mutant. The expression of NAT transcripts was analyzed by quantitative RT-PCR. Total RNA (5 μg) was used for DNase treatment and reverse transcription. Error bars indicate the technical replicates and similar results were obtained from two biological repeats.
Association of 20- to 22-nucleotide and 23- to 26-nucleotide classes of nat-siRNAs with different AGOs in Arabidopsis. Unique reads of nat-siRNAs associated with AGO1, AGO2, AGO4 and AGO7 were analyzed. Loaded reads represent unique reads of nat-siRNAs that were loaded into distinct AGOs. The percentage in total nat-siRNA-AGO libraries indicates the distribution of loaded nat-siRNAs in different AGOs.
SRO5-P5CDH siRNAs derived from salt and cold stress challenged Arabidopsis. The siRNAs were identified from GEO database accession number GSE33642. (a) Reads of siRNAs positively/negatively match to At5G62520 (SRO5) in the overlap/non-overlap region. The '+ strand' and '- strand' indicate positively or negatively matching. (b) Distribution pattern of SRO5-P5DCH siRNAs. siRNAs positively or negatively matched to At5G62520 are displayed above or below the SRO5-P5DCH gene pair. Exons and introns are represented by red and blue dishes, respectively. The overlapping region is indicated by the two green lines.
We thank David Baulcombe, Jim Carrington, Herve Vaucheret, Xuemei Chen, John Clarke and Zhixin Xie for providing seeds of various mutants. This work was supported by NIH grant R01GM093008, a NSF Career Award MCB-0642843, and an AES-CE Research Allocation Award PPA-7517H to H Jin, by NSF grant DBI-0743797, NIH grants RC1AR058681 and R01GM086412 to W Zhang, NIH grants R01GM070795 and R01GM059138 to J-K Zhu and by National Science Foundation of China (30900102) to X Zhang.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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The Prologue of the story that will spark off what is to be the biggest battle in history. Both sides are equal, but who will survive?
The Prophet
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This is a lively, exciting piece which holds its audience captive from the very first semiquaver upbeat. Judgement day prophecies are silenced by the sight of a young girl on her way to meet her... More > beloved; the prophet realizes that warnings of impending judgement cannot compete with a love which is real and present. This is the english version of the piece "Snart kysser hun sin venn i rosenhagen."< Less
The Prophet
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The Prophet is a book of 26 prose poetry essays written in English by the Lebanese artist, philosopher and writer Kahlil Gibran. It was originally published in 1923 by Alfred A. Knopf. It is Gibran's... More > best known work. The Prophet has been translated into over forty different languages. The prophet, Almustafa, has lived in the foreign city of Orphalese for 12 years and is about to board a ship which will carry him home. He is stopped by a group of people, with whom he discusses topics such as life and the human condition. The book is divided into chapters dealing with love, marriage, children, giving, eating and drinking, work, joy and sorrow, houses, clothes, buying and selling, crime and punishment, laws, freedom, reason and passion, pain, self-knowledge, teaching, friendship, talking, time, good and evil, prayer, pleasure, beauty, religion, and death. Khalil Gibran (1883–1931) was a Lebanese artist, poet, and writer. Born in the town of Bsharri in the north of modern-day Lebanon.< Less
The Prophet
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The Prophet is perhaps the most beautiful series of poetically wise musings on love, family, work, death, and all the other things that matter most, which was ever written. Its author Kahlil Gibran... More > is the third best-selling poet of all time, behind only Shakespeare and Lao-Tzu. This book has sold tens of millions of copies and is continuously on all the international bestseller lists. It has been translated into over 50 different languages and has never been out of print.< Less
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Friday, August 5, 2016
Please send us an email if you would like to receive an invitation to our 2016 olive harvest weekends, October 15-16 and/or October 22-23.
Other things to do: Swimming, hiking, creek exploration
Every family will go home with a gallon of olive oil. Every couple gets a half gallon. Every single person gets 2 500 ml bottles. You can also make arrangements to purchase additional oil, as well as our flavor infused oils and farmstead fruit vinegars. Pick as many olives as you want to take home for curing, no charge.
Good food, good friends, hard work, camping under the stars, and a tour of the Capay Valley's newest olive mill, Olica. You will see how the olives are milled into oil and do a tasting of different varietals, as well as learn about the industry and what makes an oil extra virgin.
Please join us!
Posted by Casa Rosa Farm at 5:22 PM 0 comments Links to this post
Wednesday, May 25, 2016
Roast duck!
So since we've gotten our ducks back from the processor, I've been researching duck recipes, and last night we roasted a duck. I used a method where I melted pork fat and basted it over the seasoned duck periodically while it roasted. Otherwise, I did it exactly as I do chickens- in my cast iron dutch oven in the oven.
Fool proof Roast Duck
1 duck, about 3-4 lbs
fresh herbs, chopped (I used cilantro, oregano and basil)
salt, coarse sea salt or Redmond salt preferred
Pork fat, or olive oil, about 1 cup
Let sit in the refrigerator overnight or for 4 hours.
Lay duck in a dutch oven (I have a cast iron one with enamel coating) breast down. Set oven temp at 325 degrees. Put cover on. Bake for about 1 hour. Baste with fat (use the duck fat in the pan or melted lard if there isn't enough) Depending on the weight of the duck, you'll bake it for about 2-3 hours. For the last 45 minutes, remove cover and turn duck breast side up to crisp the skin. I decided our duck was done when the leg pulled away easily from the body and the skin looked brown and lacquered.
Fool proof Roast Duck
1 duck, about 3-4 lbs
fresh herbs, chopped (I used cilantro, oregano and basil)
salt, coarse sea salt or Redmond salt preferred
Pork fat, or olive oil, about 1 cup
Let sit in the refrigerator overnight or for 4 hours.
Lay duck in a dutch oven (I have a cast iron one with enamel coating) breast down. Set oven temp at 325 degrees. Put cover on. Bake for about 1 hour. Baste with fat (use the duck fat in the pan or melted lard if there isn't enough) Depending on the weight of the duck, you'll bake it for about 2-3 hours. For the last 45 minutes, remove cover and turn duck breast side up to crisp the skin. I decided our duck was done when the leg pulled away easily from the body and the skin looked brown and lacquered.
Posted by Casa Rosa Farm at 9:27 PM 0 comments Links to this post
Sunday, May 8, 2016
Regenerative Grazing
Below is a pic of a grazing field section that has very poor soil. We concentrated all of last years hay feeding onto this area, leaving a thick layer of mulch to rot over winter. Last May, after grazing, it was mostly star thistle and medusa grass (both invasive plants that cattle only eat when young). This year, after grazing, there are very few star thistle shoots, and medusa heading has been reduced by well over 50%. We also have better regrowth of the native ryes & bromes, as well as the oats & barley that were introduced by the feeding hay on the ground. Our hope is to graze this area a third time (thanks, extra 2016 rain!) with our sheep, who generally eat star thistle while green but before it heads out.
|Grazed regrowth of hay mulched area
Next is a 3 acre section of wheat/barley nurse crop for alfalfa that was cut to windrows. Although our cows did a great job cleaning up the rest of the field, they could not finish off this section before we had to start irrigating. At first we were very worried that we'd lose the alfalfa that we had planted as an under story crop where each windrow was left. However, what we are finding is that's not the case. Well established alfalfa will regrow though a windrow mulch, but this is new alfalfa. It hasn't been cut yet, ever.
|Whole 3 acre section, with windrows
|Alfalfa with no windrow mulch cover
|Alfalfa under windrow, mulch pulled aside
It's Alive!
Posted by Casa Rosa Farm at 6:02 PM 0 comments Links to this post
Wednesday, April 13, 2016
Some big happenings this spring!
DucksWe're raising 400 pekin ducks on pasture. Our latest poultry housing is similar to a hog hoophouse, but in California, we have no need for shelter from the rain, only the sun. The panels are covered with a layer of shadecloth which doubles as predator barrier.
Pekin ducks are ready for processing in about 4 months on pasture. Expect to see whole frozen ducks at market and at retail outlets by June. We're enjoying a break from broiler chickens, but we're getting back to those from August to October.
|Duck Schooner #3
We're having an open farm day!
Come learn about rotational grazing, double cropping (orchards and animals) see our 100% solar irrigated hay field in action, and learn about sustainable meat production, while enjoying a bbq, baby animals for the kids, and the beautiful Capay Valley.
Date: April 30th. Contact us for more information and to register.
|Steers grazing
We're starting a Thursday farmer's market in San Francisco!
Come see us in SF Mission District at the Mission Community Market on 22nd and Bartlett, Thursdays from 3-8 pm.
This is a fun community market with a lot of great vendors such as Blue House Farm, Happy Boy, Yerena Farms and more. We'll be selling all our products: beef, lamb, pork, eggs, olive oils, olive oil body care products, herbs, jams and more.
Check out the market at: www.missioncommunitymarket.com, or on instagram at @missioncommunitymarket or on facebook.
As a special introductory offer, we are giving 50% off every bottle of olive oil purchased at our first market. Come make us feel welcome!
Posted by Casa Rosa Farm at 2:17 PM 0 comments Links to this post
Monday, December 14, 2015
With an oink oink here and an oink oink there, here we grow!
Pastured pork is our newest foray, and unlike broiler chickens, or geese, this one is here to stay.
We've just picked up 2015's pork- 10 delicious hogs are now in the freezers, partitioned out into ham, bacon, pork chops and sausage.
We've got our three Berkshire/GOC/Hampshire sows: JLo, Houdina, and Lucy, and our upstanding Berkshire boar, Quasimodo established in their farrowing kingdom, and a new set of 20 feeder piglets ready to go out on pasture this spring.
Our decision to raise pork comes from our desire to be as well rounded a farm eco-system as possible, and there is so many things on a small farm a pig can eat that go to waste otherwise. They are also quick to finish- taking about 1/3 of the time of a grass finished beef and 3 months less than a grass finished lamb. Throughout the summer and fall, our first batch of 10 pigs were rotated through a series of pastures within the orchards and also through pastures where the sheep had been overwintered, and they plowed up and ate tons of grubs and bugs, weeds, roots and even... grass. They love clover and alfalfa and will dig the whole plant up and eat it. I also fed them dropped fruit of all kinds from our orchard- apricots, pears, plums, crab apples (which even the chickens don't like) persimmons, quince. They also did a great job taking care of any overripe melons in my summer melon experiment. And when I was done with the melons, I fenced it in with electric net and turned the hogs out to root out all the remaining melon plants, melons, sunflowers, amaranth and sorghum I had inter-planted. They dug up the sunflower stalks and ate the roots. They even made some inroads on the bindweed and bermuda grass rhizomes, but I would have had to irrigate the field fairly well before turning them in for it to have had a lasting effect. Right before slaughter, they were eating acorns and pecans and black walnuts along with pomegranates and olive pomace from our olive oil milling.
Pigs are fun to have on the farm! They are playful and enjoy getting their bellies scratched.
In other news, our 100% solar irrigated field is finally taking off. The frost has knocked back some of the alfalfa germination, but the wheat nurse crop is coming up well with the rains. In our other field where no irrigation is available, we have seeded the last of our dryland pasture mix, as well as our traditional oat/wheat/barley/vetch hay planted on our parent's rented ground. It was a bit nerve wracking there for a while with too little rain to actually support the crop, but enough moisture to germinate, then wilt it. With last weekend's storm, we should now be in good shape, until mid January 2016. Hopeful (with lots of fencing work) we can have all the animals out on pasture this year for the majority of the growing season.
Even with the drought, more regulations and less irrigation water available than ever before for farming in California, we continue to grow our business. 2015 has been a year where we look back and recognize that we have been direct selling quality grass fed meats since 2010 and have been farming together as a family for 7 years. We hired our first employees this year and continue to welcome the occasional HelpX and WWWOOF visitor to our farm. We also continue to perfect the products we produce and pride ourselves on our innovative use of a carcass to give modern urban customers the cuts and portions they are looking for. Our partnerships with Manas Meats here in the Capay Valley and Roundman's Smokehouse in Ft. Bragg ensure that our quality pasture raised and grass fed meat is enhanced by artisan smoking and sausage making, with a strong emphasis on traditional recipes and natural curing methods without the use of added nitrates. If you haven't tried our pork bacon, sausage, summer sausage, sliced ham, pastrami or our innovative beef bacon, please do.
Posted by Casa Rosa Farm at 4:37 PM 0 comments Links to this post
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Love this hotel! Stayed here first time trip to the gulf..beautiful view beautiful room!! Signed the papers on our new residence here!! Love it!!!! The staff was wonderful..food and drinks perfect. We upgraded to a corner room on the 7th floor and the view brought me to tears..I am a California native but give me the gulf anytime, so much prettier and layer back...recommend this this resort to anyone..clean.
- Official Description (provided by the hotel):
- DiamondHead Beach Resort & Spa is the premier choice for Fort Myers Beach lodging. It offers 121 one-bedroom suites with a king-size plush pillow top bed, private bath, living room with queen-size sleeper sofa and a fully stocked kitchenette. WiFi internet connection throughout the resort. Enjoy casual dining right on the beach at Cabana's Beach Bar & Grille and upscale dining at Chloes and Chloes Lounge. Amenities include The Esterra Spa & Salon, watersports, two outdoor hot-tubs, heated Gulf-side pool, exercise room, piano bar, private screened balconies and spectacular Gulf of Mexico sunsets. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Expedia, Booking.com, TravelClick, Hotwire, Orbitz, Odigeo, HotelTravel.com, Hotels.com and getaroom.com so you can book your DiamondHead Beach Resort & Spa reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Diamondhead Beach Hotel Fort Myers Beach
- Diamondhead Beach Resort
- Diamondhead Resort In Fort Myers Fl
- Diamond Head Beach Resort


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Saturday, August 8, 2009
Investigating the Irish Diaspora in Photos
The Irish Diaspora in Philadelphia, Part I
Irish music and dance was big in Philadelphia long before Sinead O'Connor shaved her head and became a priest or Michael Flatley stepdanced his way to numerous lawsuits. Keep your eyes open for the historical proof of my assertions.
The photos in this slide show are in three categories -- first, a trip to the Historical Society of Pennsylvania to dig into the collection from the now defunct Balch Institute for Ethic Studies. After walking around the lovely building designed by Quaker architect Addison Hutton, which was built in 1902, I settled into the Robin Hiteshaw collection, and found old flyers from Irish-American dances, benefits and applications to benevolent societies. Great fun could be had for 15 cents at one time in Philadelphia's Irish-American community!
Next, I walked down Juniper to Drury, where my brother Steve joined me at McGillin's Olde Ale House, which was founded by two Irish immigrants in 1860, and is now in a countdown to its 150th anniversary. Though Steve spent many a happy hour at McGillin's in the past, he didn't know that it is an ancestral watering hole of the Allen clan, having been frequented by our late grandfather. Sláinte, Steve!
Finally, a friend joined me to vist The Irish Memorial, located at Front and Chesnut Streets on Penn's Landing, in view of the Benjamin Franklin Bridge. This memorial by Glenna Goodacre commemorates the Irish Potato Famine, which began in 1840, and lasted through 1846. It caused the deaths of one million Irish citizens, and created a massive migration to America. The memorial features 35 individual bronze statues that depict the hopelessness and desperation of life in Ireland during the Famine; the ordeal of the ocean voyage to Philadelphia and other American ports; and the hopeful arrival on the shores of the Delaware River.
Someone I met at McGillin's told me Sinead and the Lord of the Dance himself modeled for the Memorial.
Mere blarney I did not investigate.
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Look at this gorgeous fella! This blue fawn stunner was born on 7-22 and will be ready to go on 9-16. He has been handled, loved on and spoiled since day one. He will have 2 sets of shots and several wormings. Mom is a fawnequin and dad is blue. We are located near Elkins, Wv, but we can meet part way for an additional fee.
Aug 30 -
Harrisonburg :
Mill Creek - Dogs & puppies
Gorgeous Blue and Black Great Dane Puppies
8 weeks old - ready to go home ( July 4th )
4 blue males , 2 black males and 1 back female available
Raised in the home with kids cats and other dogs
Very confident, social puppies
House trained
Micro-chipped, AKC/CKC registered , dewormed ( interceptor plus)
Health certificate , Distemper vaccine.
Facebook: gentle giants MA
Life time ...
Aug 29 -
Brattleboro :
Athol - Dogs & puppies
We have 3 AKC REGISTERED great Dane puppies available September 3rd.Health certificate,vet checked,first set of shots and dewormed.pre-spoiled,raised around other dogs and small children.mom is a blue and dad is a Harlequin.both on premises.
Aug 28 -
Harrisburg :
Williamsport - Dogs & puppies
Our beautiful puppies are almost 8 weeks old ( 29th August )
Our mothers ( Twyla and Mistey ) are imported from South Africa.
Father is a championship bloodline.
We have 4 blue males, 2 black males and 1 black female. They have all been raised in our home with our kids ( ages 4-9), cats and other dogs, registered, micro-chipped, vaccinated, dewormed and doing well with their house ...
Aug 26 -
Brattleboro :
Phillipston - Dogs & puppies
Hello, I want to tell you all my babies were born August 22,2016. I have 4 Harlquion, 2 black and one blue merle.. This what is still available. Let mom do what she has too. Let the babies nurse and grow. Any one interested contact me leave name address and phone number so I can send pictures when the time is right. To young right now in a few days, will get back to you.
800.00 with out ...
Aug 21 -
Lynchburg :
Meherrin - Dogs & puppies
Gorgeous Great Dane Puppies. So gentle and affectionate. I have 3 left . This is a great breed for families with children. They are very easy to train and eager to please. Puppy is now ready to go to new homes as they have just received their first shots and de worming.for more info text...(619) 348-5098
Aug 21 -
Philadelphia :
Morrisville - Dogs & puppies
I have 1 black male $800, 1 Merle female with a short tail $900, 1 heavy marked harlequin male $1,000 and 1 light marked harlequin male (pick of litter) $1,200. They will come with there Akc paperwork, wormed and first vaccinations. Ready to go next week.
Aug 17 -
Pittsburgh :
Mount Pleasant - Dogs & puppies
AKC reg puppies harlequin and merles pick your new best friend today...
Aug 16 -
Harrisburg :
Turbotville - Dogs & puppies
Blue Great Dane puppies ,
AKC / CKC / UKC registered
. Raised with kids , cats and other dogs, parvo vaccine, microchipped .
Distemper Vaccine
Only to the best homes $1900
Ready to leave 29th August
Aug 16 -
Brattleboro :
Phillipston - Dogs & puppies
Female great Dane pup. Born 6/19...ready to go week of 8/14... dam (160lbs) is a Merle, sire (160lbs) is a black. Pups are 68% euro. $500 deposit
Puppies will go home with:
Health certificate
First shots
Age appropriate dewormings
2 yr health warranty
Lifetime breeder support
Akc papers are available for extra $200
Aug 15 -
Boston :
Leominster - Dogs & puppies
Female great Dane pup Born 6/19...ready to go week of 8/14... dam (160lbs) is a Merle, sire (160lbs) is a black. Pups are 68% euro. $500 deposit
Puppies will go home with:
Health certificate
First shots
Age appropriate dewormings
2 yr health warranty
Lifetime breeder support
Akc papers are available for extra $200
Aug 11 -
Boston :
Leominster - Dogs & puppies
Gorgeous solid blue male. He is being offered with full AKC registration. He is UTD on shots and DNA on record. He just sired a beautiful litter of 18 puppies!!! I am no longer going to be breeding Great Danes so this would be a good opportunity for someone. He has always been in a climate controlled kennel with a huge outdoor run. Call or email with any questions or to come visit Triton. ...
Aug 4 -
Delaware :
Delmar - Dogs & puppies
Male Harlequin Great Dane Puppy ready to go end of august
Aug 2 -
Pittsburgh :
Mount Pleasant - Dogs & puppies
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Tiësto Spotted at Santa's Enchanted Forest's Techno Power
There are a lot of things we expect to encounter when going to Tropical Park in west Miami-Dade for Santa's Enchanted Forest: chongas, carnies, awesome carnival food. But we definitely don't expect to see a superstar DJ.
Nevertheless, we spotted Tiësto there just the other day. Of course, not in person ... More like a mildy creepy version of the Dutch DJ airbrushed on the enormous display behind the Techno Power ride.
But the cherry on top is the ride itself. Our intrepid photographer, Ciara Osorio, who spotted the cartoonish Tiësto, reported that while riders are being whirled around, the soundtrack is anything but techno. "It's all country music."
See the cut for a few more photos of Tiësto at Santa's Enchanted Forest.
Photo by Ciara Osorio
Photo by Ciara Osorio
Photo by Ciara Osorio
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HourlyMore than 30 hrs/week
Entry Level
Start DateSeptember 25, 2013
These instruction are very in depth, but an online demo can be given if needed.
Your job is to analyze data through the Radius app and then add them into a spreadsheet.
Radius is a database of over 26 million small businesses in the US. It is a user friendly app for creating lead lists and analyzing data on small businesses.
Before you log into the app, create an excel spreadsheet. You will vertically record the 100 cities given in this URL into the spreadsheet: http://www.city-data.com/top1.html
You will create 4 horizontal columns which will be labeled "Has Website," "Has Facebook," "Has Yelp," and "Has Foursquare." After you create this spreadsheet you will start recording the data from the app. The steps for extracting the data from the app are below:
Step 1. Log into Radius (we will provide you with username and password)
Step 2. Select the upper left button "create a lead list"
Step 3. Scroll to the industry section and select restaurants (you will never change the industry as we are only analyzing the restaurant industry in the US)
Step 4. Scroll to the location section and select one of the cities from the list given. At the top of the window you should see a number next to the field for "name your list." (record the number as this will be the total number of restaurants in a given city)
Step 5. Scroll to web and social activity and select yes to "has an official website." A new number should appear next to the "name your list" field. (record the new number as it will be the number of restaurants with a website in the given location)
Step 6. Unclick the "has an official website" button and click the yes button for "has a Facebook page." (record the new number as it will be the number of restaurants in the selected area with a Facebook page).
Step 7. Unclick the "has a Facebook page" button and click the yes button for "has a Yelp page." (record the new number as it will be the number of restaurants in the selected area with a Yelp page).
Step 8. Unclick the "has a Yelp page" button and click yes for "has a foursquare page." (record the new number as it will be the number of restaurants in the selected area with a Foursquare).
Step 9. Unclick the "has a Foursquare" button.
Step 10. You now should have only the location and industry selected. Return to location and unclick the location you currently have selected. Then find the next location on your list and click it. (Record this number as it will be the number of restaurants in your next location).
Step 11. Start process over again from step 5
You will record this data for each city. After a while this will make perfect sense and will not take much thought. It is not difficult work at all, it is merely time consuming. You are never adjusting the industry section. You are analyzing data for each city based off the restaurant industry and the web activity of restaurants in that area.
Like I stated above, a demo can be provided if necessary.
Activity on this Job
Last Viewed by Client: 2 years ago
Hired: 1
About the Client
United States
San Francisco 10:21 AM
Jobs Posted
50% Hire Rate, 1 Open Job
Total Spent
4 Hires, 1 Active
Avg Hourly Rate Paid
29 Hours


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Boxer & Siberian Husky mix ,female 8 weeks old. 2 sets of shots, tail is docked. Loves kids and other pets ,loves to play ,pretty blue eyes.Needs a new home now will consider trades
Aug 12 -
Cincinnati :
Hillsboro - Dogs & puppies
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DENVER -- Barreling around the corner like a pulling guard, Peyton Manning cleared the way for an extra five yards.
So maybe it wasn't the best idea to have the quarterback as the lead blocker in a preseason game, but Manning wasn't about to let up.
Manning had two touchdown passes in the first half, then threw a key block to set up another score as the Indianapolis Colts beat the Denver Broncos 28-23 Monday night.
"We're playing on national television and I don't care if it is the preseason," said Manning, who finished 15-for-22 for 157 yards. "You want try to send some kind of message to the other teams and to the country that we feel like we're going to have a good team this year."
Recent injuries to quarterbacks Michael Vick and Chad Pennington had some bemoaning that the preseason is too long, but Manning played as if it was a playoff game.
He threw touchdown passes to Dallas Clark and Brian Allen in the first half, then was the lead blocker on a 16-yard double reverse by Marvin Harrison on the Colts' first drive of the third quarter.
Manning got in several good shots on Denver cornerback Kelly Herndon on the reverse before walking back to the huddle with a big smile on his face.
Allen scored on a 4-yard run six plays later, stiff-arming Sam Brandon at the line to put the Colts (2-1) up 21-13 with 4:49 left in the third quarter.
"You can't worry about that. You have to go play," Colts coach Tony Dungy said of potential injuries to his blocking quarterback. "Usually, when you're playing hard, good things happen for you."
Denver quarterback Jake Plummer got the playing hard part right, but that didn't stop him from being booed in his first home game with the Broncos.
Plummer fumbled his first snap, threw a pass that Colts linebacker Gary Brackett should have intercepted, and missed a wide-open Ed McCaffrey on what would have been an 18-yard touchdown. And that was just on the first drive.
"I don't think he was nervous," Broncos coach Mike Shanahan said. "I just think he was like everybody else - looking forward to playing the game."
Plummer was much better on Denver's second drive, thanks to Ashley Lelie.
Plummer rolled out on the first play of the series, thought about running, then pulled up for a pass at the last second. Lelie adjusted to the slightly underthrown ball and made a leaping grab over Mike Doss for a 55-yard touchdown.
But that was about it for Plummer.
He dropped another snap in the second quarter, then was booed after Anthony Floyd intercepted his overthrown pass on Denver's first drive of the third period.
It was his final pass before Steve Beuerlein took over midway through the third quarter.
"I'm not too concerned," said Plummer, who was 15-for-22 for 205 yards. "Things will improve. I'm not happy with the way it ended. Nobody wants to throw a pick on their last throw."
The Colts sputtered early before getting things going late in the first quarter. After managing just 27 yards on its first two drives, Indianapolis moved 66 yards in nine plays for its first score. Manning capped the drive with a 14-yard pass to Clark, who was wide open after no one covered the middle for the Broncos (2-1).
The Colts didn't take long to score again.
Allen took a screen pass from Manning on the third play of the next drive, got a key block from guard Rick Demulling and turned it into a 53-yard touchdown.
Demulling took out safety Kenoy Kennedy near the 20-yard line, and Allen leaped over the corner pylon from about four yards out to put the Colts up 14-10.
"We played like we had been off 10 or 12 days, and it took us a while to get back the groove," Dungy said. "I like the way we bounced back."
Indianapolis' Jim Kubiak hit Aaron Moorehead on a 9-yard touchdown pass with 2:38 left. Denver cut the lead to 28-23 on a 5-yard pass from Danny Kanell to Dwayne Carswell with 40 seconds left, but the Colts recovered the ensuing on-side kick.
- Indianapolis backup QB Brock Huard missed his third straight game with a broken index finger on his throwing hand.
- The teams will play again Dec. 21 in Indianapolis.
- Denver LT Ephraim Salaam played in the preseason for the first time after having arthroscopic surgery on his right knee Aug. 1.
- Denver's Jason Elam hit field goals of 36, 39 and 42 yards.
- Broncos RB Clinton Portis didn't play in the second half because of a bruised calf, but the injury wasn't considered serious.
The Associated Press News Service
Copyright 2003, The Associated Press, All Rights Reserved


text:
Bridgewater, NJUSA
Hometown: Bridgewater
November 25, 2013
general interest in what's going on around our area
software development, startups, excitement, fun.
I've launched a few startups in the past. Currently involved with startups now.
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