A pathogen-inducible endogenous siRNA
in plant immunity
Surekha Katiyar-Agarwal*, Rebekah Morgan*, Douglas Dahlbeck†, Omar Borsani‡, Andy Villegas, Jr.*, Jian-Kang Zhu‡,
Brian J. Staskawicz†§, and Hailing Jin*§
Departments of *Plant Pathology and‡Botany and Plant Sciences, Center for Plant Cell Biology and Institute for Integrative Genome Biology,
University of California, Riverside, CA 92521; and†Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720
Contributed by Brian J. Staskawicz, September 19, 2006
RNA interference, mediated by small interfering RNAs (siRNAs), is
a conserved regulatory process that has evolved as an antiviral
defense mechanism in plants and animals. It is not known whether
host cells also use siRNAs as an antibacterial defense mechanism in
eukaryotes. Here, we report the discovery of an endogenous
pathogen Pseudomonas syringae carrying effector avrRpt2. We
HEN1, RDR6, NRPD1A, and SGS3. Its induction also depends on the
cognate host disease resistance gene RPS2 and the NDR1 gene that
is required for RPS2-specified resistance. This siRNA contributes to
RPS2-mediated race-specific disease resistance by repressing PPRL,
a putative negative regulator of the RPS2 resistance pathway.
antibacterial defense ? DCL1 ? RDR6 ? RPS2-specific
eukaryotic gene expression by guiding mRNA cleavage, trans-
lational inhibition, or chromatin modification (1, 2). In Arabi-
dopsis, ?100 miRNAs have been reported and shown to be
important for plant development (3, 4) and abiotic stress toler-
ance (5–7). One miRNA was recently shown to contribute to
basal defense against bacteria by regulating auxin signaling (8).
In contrast to the relatively limited number of miRNAs, thou-
sands of endogenous siRNAs have been sequenced (6, 9–11).
However, their biological roles are largely unknown except for
the functions of transacting siRNAs (ta-siRNA) in plant devel-
opment and hormone signaling (4) and the roles of some
chromatin-associated siRNAs in DNA methylation and tran-
scriptional gene silencing (4). Borsani et al. (12) recently dis-
covered a new class of endogenous siRNAs derived from the
overlapping region of a pair of natural antisense transcripts
(NATs) (12). These so-called nat-siRNAs regulate salt stress
response in Arabidopsis (12). Despite large intergenic spaces, a
significant proportion of eukaryotic genomes are arranged as
NATs (13). More than 1,000 pairs of NATs exist in Arabidopsis
(14, 15). Our analysis of transcript profiles from an Arabidopsis
microarray database (16) has revealed that, in many cases, one
transcript of a NAT pair is specifically induced under certain
abiotic or biotic conditions. The induced transcript may pair with
the existing antisense transcript and trigger the nat-siRNA
formation, resulting in the silencing of the antisense transcript in
cis or other homologous loci in trans. NATs may serve as one of
the major sources of endogenous siRNAs for gene regulation in
response to different environmental conditions. This hypothesis
is well supported by the presence of ?100 nat-siRNAs in the
Massively Parallel Signature Sequencing (MPSS) and Arabidop-
sis Small RNA Project (ASRP) databases (9, 17). In this study,
we identified a nat-siRNA that is specifically induced by the
bacterial pathogen Pseudomonas syringae (Ps) carrying effector
avrRpt2 (18). We demonstrate that its induction depends on a
novel biogenesis pathway that requires the cognate host disease
resistance (R) gene RPS2 (19) and the NDR1 (20) gene that is
ndogenous small interfering RNAs (siRNAs) and microR-
NAs (miRNAs) have emerged as important regulators of
also required for RPS2-specified resistance. This siRNA re-
presses a negative regulator of the RPS2 resistance pathway.
Induction of a nat-siRNA by Bacterial Pathogen Ps Carrying avrRpt2.
Pathogen effectors can be recognized by R proteins and can
trigger a series of disease resistance responses, including acti-
vating and repressing a large array of genes (21). To address
whether endogenous siRNAs play a role in gene expression
reprogramming in R gene-mediated disease resistance, we
searched the small RNA databases (9, 17) and examined nat-
siRNAs generated from NAT pairs that are potentially regulated
by bacterial pathogenesis. Excitingly, we discovered that a 22-nt
nat-siRNA (ASRP1957), derived from the overlapping region of
a Rab2-like small GTP-binding protein gene (ATGB2,
At4g35860) and a PPR (pentatricopeptide repeats) protein-like
gene (PPRL, At4g35850), is strongly induced by Ps pathovar
tomato (Pst) carrying avirulence (avr) gene avrRpt2 but not
avrRpm1, avrRps4, or avrPphB (Fig. 1A). We named it nat-
siRNAATGB2. We used Pst strain DC3000 for all of the
experiments in this study.
The nat-siRNAATGB2 sequence is complementary to the 3?
UTR region of the antisense gene PPRL and thus could poten-
tially induce silencing of PPRL. We examined the expression of
PPRL as well as the sense gene ATGB2 upon Pst challenge. The
ATGB2 transcript was strongly induced by both Pst (avrRpt2) and
Pst (avrRpm1) (Fig. 1B), whereas the PPRL mRNA was sub-
stantially down regulated only by Pst (avrRpt2) infection where
the nat-siRNAATGB2 was strongly induced (Fig. 1 A and C).
The result suggests that down-regulation of PPRL depends on
the induction of the nat-siRNAATGB2 and the induction of
ATGB2 alone is not sufficient for inducing nat-siRNAATGB2.
To determine whether the induction of ATGB2 is necessary for
inducing nat-siRNAATGB2, we obtained a T-DNA insertion
line (Salk?083103) of ATGB2 from the Salk collection (22). The
homozygous line is a partial knock-down mutant with T-DNA
inserted in the 3rd intron (Fig. 1D). We detected less induction
of nat-siRNAATGB2 and less repression of PPRL mRNA
expression in this knock-down line than in the WT plants after
Pst (avrRpt2) challenge (Fig. 1 D and E). These results suggest
that the induction of sense transcript ATGB2 is necessary but not
sufficient for nat-siRNAATGB2 accumulation (Fig. 1 A, B, and
D), implying that the induction of this siRNA is under multiple
Author contributions: H.J. designed research; S.K.-A., R.M., D.D., O.B., A.V., and H.J.
performed research; J.-K.Z., B.J.S., and H.J. analyzed data; and S.K.-A. and H.J. wrote the
The authors declare no conflict of interest.
Abbreviations: miRNA, microRNA; ta-siRNA, transacting siRNA; NAT, natural antisense
transcript; Ps, Pseudomonas syringae; Pst, Ps pathovar tomato; avr, avirulence; DCL,
DICER-like; RDR, RNA-dependent RNA polymerase; Dex, dexamethasone; hpi, h postinocu-
lation; dpi, days postinoculation; PPR, pentatricopeptide repeat; PPRL, PPR protein-like;
HR, hypersensitive responses.
§To whom correspondence may be addressed. E-mail: email@example.com or stask@nature.
© 2006 by The National Academy of Sciences of the USA
November 21, 2006 ?
vol. 103 ?
layers of control and requires other factors associated with Pst
Biogenesis of nat-siRNAATGB2.To define the components required
for its biogenesis, we examined nat-siRNAATGB2 in Pst (avr-
Rpt2)-challenged small RNA biogenesis mutants and the corre-
sponding WT ecotypes. The Arabidopsis genome has four DI-
CER-like (DCL) proteins (23). Interestingly, the induction of
nat-siRNAATGB2 could be detected in dcl2-1, dcl3-1, and
dcl4-2 mutants but not in the dcl1-9 mutant (Fig. 2A). The result
indicates that the miRNA biogenesis component DCL1 is re-
quired for the formation of nat-siRNAATGB2. This observation
differs from the biogenesis of the 24-nt nat-siRNASRO5 that
requires DCL2, 21-nt nat-siRNAs that require both DCL1 and
DCL2 (12), and ta-siRNAs that require DCL1 and DCL4 (24).
We did not detect any other siRNAs generated from the
overlapping region of PPRL and ATGB2 or siRNA that is
complementary to nat-siRNAATGB2 (data not shown). Thus,
this nat-siRNA is generated from a specific site of the overlap-
ping region of PPRL and ATGB2 transcripts and is strand-
Mutations in the dsRNA-binding protein HYL1 and RNA-
dependent RNA polymerase (RDR) 6 also totally blocked the
accumulation of nat-siRNAATGB2, whereas a mutation in
RDR2 had no effect (Fig. 2A). RDR6 is required for virus-
induced gene silencing, transgene silencing, and ta-siRNA pro-
duction (25). HYL1 has been indicated to interact with DCL1
(26) and affects the accumulation of several miRNAs (27) and
ta-siRNAs (24). The level of nat-siRNAATGB2 was reduced in
sgs3, the RNA methyltransferase mutant hen1, and the RNA
polymerase IVa mutant nrpd1a (Fig. 2A), which is similar to that
of salt-induced nat-siRNAs (12). These results suggest a biogen-
esis pathway for nat-siRNAATGB2 in which nat-siRNAATGB2
is processed by the DCL1-HYL1 complex, stabilized by HEN1-
mediated methylation, and amplified by RDR6-, SGS3-, and
RNA polymerase IVa-mediated reactions.
To confirm that the down-regulation of PPRL depends on the
induction of nat-siRNAATGB2, we examined the PPRL mRNA
level in the mutants that failed to generate nat-siRNAATGB2
upon Pst (avrRpt2) infection. The down-regulation of PPRL was
abolished in dcl1-9, hyl1, and rdr6 compared with WT Landsberg
erecta (Ler), Nossen-0 (No) and C24, respectively (Fig. 2B). The
sgs3 and nrpd1a mutants, where the accumulation of nat-
siRNAATGB2 is significantly reduced, also accumulate 2- to
3-fold more PPRL mRNA than that in their corresponding
controls (Fig. 2B). Thus, the down-regulation of PPRL is me-
diated by nat-siRNAATGB2.
To test whether the overlapping region is sufficient for gen-
erating nat-siRNAATGB2, the full-length or overlapping region
of ATGB2 was coexpressed with PPRL transiently in Nicotiana
benthamiana leaves. Flag-tagged PPRL cDNA with its 3? UTR
was cloned into a binary vector driven by the CaMV 35S
promoter. Full-length (F) or only the overlapping region (O) of
ATGB2 cDNA was cloned into the inducible expression vector
Small RNA was extracted from the leaves harvested at 15 hpi of Pst (2 ? 107cfu?ml) carrying various avr genes and an oligonucleotide probe complementary
to the siRNA was used. DNA probe was used for detecting U6 RNA for measuring the relative abundance (RA) (shown below). Ethidium bromide-stained tRNA
is also shown as a loading control. (B and C) Relative expression levels of ATGB2 (1B) and PPRL (1C) as measured by real-time RT-PCR. The expression levels of
AtGB2 and PPRL were normalized to that of ubiquitin. (D) Northern blot analysis shows reduced ATGB2 and nat-siRNAATGB2 expression levels in Salk?083103
homozygous line upon Pst (avrRpt2) (2 ? 107cfu?ml) challenge comparing to the WT Col-0. The levels of actin and U6 were used for quantification and loading
were plotted from three replicates (B, C, and E).
A nat-siRNA is induced by Pst (avrRpt2). (A) Detection of the nat-siRNA by Northern blot analysis. The nat-siRNA sequence is shown under the panel.
Katiyar-Agarwal et al. PNAS ?
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pTA7002 (28). Substantial down-regulation of PPRL was ob-
served at both the RNA and protein levels after induction of
either full length (F) or only the overlapping region of ATGB2
(O) by dexamethasone (Dex) (Fig. 2C), as was the induction of
nat-siRNAATGB2. The results suggest that the overlapping
region alone is sufficient to give rise to the nat-siRNA and to
induce antisense gene silencing.
Induction of nat-siRNAATGB2 Depends on RPS2 and NDR1. Pathogen-
derived effectors are recognized directly or indirectly by specific
plant R proteins and trigger rapid race-specific resistance re-
sponses. The effector avrRpt2 of Ps is specifically recognized by
the coiled-coil NBS-LRR type R protein RPS2 (19) and triggers
a series of resistance responses, including the generation of
reactive oxygen species, reprogramming of gene expression, and
induction of hypersensitive responses (HR), which limit bacterial
growth. The specific induction of nat-siRNAATGB2 by avrRpt2
race-specific disease resistance. To further understand the reg-
ulation of nat-siRNAATGB2 by pathogen infection, we exam-
nat-siRNAATGB2 (A) and ATGB2 (B) was performed on Pst (avrRpt2)-treated defense-signaling mutants and WT Col-0 plants. U6 RNA was used for small RNA
quantification. (C) Relative quantification of PPRL expression in defense signaling mutants by real-time RT-PCR analysis. PPRL expression level was normalized to that
of ubiquitin. The expression levels in untreated WT Col-0 were used as 100%. Standard deviations were plotted from three replicates.
Accumulation of nat-siRNAATGB2 is controlled by RPS2 and some components of the disease resistance signaling pathway. Northern blot analysis of
nat-siRNAATGB2 in various Pst (avrRpt2)-treated small RNA biogenesis mutants and their corresponding WT controls. MiR171 and U6 RNA was used as controls.
U6 level was used for quantification. (B) Relative PPRL mRNA levels in sgs3, dcl1-9, hyl1, rdr6, nrpd1a, and their corresponding WT controls after Pst (avrRpt2)
infection. The expression levels were normalized to that of ubiquitin. The expression level in untreated WT Col-0 was used as 100%. Standard deviations were
plotted from three replicates. (C) Transient coexpression of PPRL and ATGB2 in N. benthamiana. Agrobacterium GV3101 harboring PPRL was coinfiltrated with
GV3101 carrying full-length (F) or only the overlapping region (O) of ATGB2 constructs into 3-week-old N. benthamiana leaves. The expression of AtGB2 was
from a gel that was run in parallel was stained with Coomassie blue for Western blot loading control. nat-siRNAATGB2 is detected after the induction of either
full-length or overlapping region of ATGB2. (Bottom) tRNA was used as a loading control.
Accumulation of nat-siRNAATGB2 depends on DCL1, HYL1, and RDR6, and also requires HEN1, NRPD1a, and SGS3. (A) Northern blot analysis of
www.pnas.org?cgi?doi?10.1073?pnas.0608258103Katiyar-Agarwal et al.
ined its accumulation in various mutants of resistance signaling
components. Although the induction level of ATGB2 by avrRpt2
was not substantially different in these mutants, the accumula-
nat-siRNAATGB2 was not detected in rps2 (101C) and ndr1
mutants, which indicates that nat-siRNAATGB2 induction re-
quires the functional resistance protein RPS2 and NDR1, both
of which are required for avrRpt2-induced resistance (20). Mu-
tations in other resistance signaling components, SCF ubiquitin
ligase complex component SGT1b, systemic acquired resistance
signaling component NPR1, and ethylene signaling component
EIN2 also reduced the level of nat-siRNAATGB2. Consistent
with the silencing of PPRL by nat-siRNAATGB2, the mutants
with no or reduced levels of nat-siRNAATGB2 showed no or
less suppression of PPRL expression (Fig. 3C). These mutations
had no effect on the accumulation of miR173, which demon-
strates a specific biogenesis regulation of nat-siRNAATGB2 by
the disease resistance signaling pathways. The jasmonic acid
(JA) signaling mutant jar1, salicylic acid (SA) signaling mutant
pad4, SA biosynthesis mutant eds16, and SGT1b homologue
SGT1a had no effect on nat-siRNAATGB2 accumulation (Fig.
3A). These results suggest that some components in basal
defense and ethylene signaling may interfere with nat-
on the down-regulation of PPRL by nat-siRNAATGB2 in re-
sponse to Pst (avrRpt2) challenge, we hypothesized that PPRL
may negatively regulate RPS2-mediated resistance. PPRL is an
atypical PPR protein with an unknown function and is localized
in mitochondria (29). To assess its function in disease resistance,
we isolated T-DNA insertion lines of PPRL (Salk?013843 and
Salk?071137) from the Salk collection (22) and also generated
PPRL cDNA-Flag (without UTR) overexpression lines (Fig. 4A)
for loss- and gain-of-function studies. Complete knockout of
PPRL expression may lead to enhanced disease resistance to
avrRpt2 because the PPRL gene is silenced after pathogen
infection in the WT resistance plants. No difference was ob-
served in the growth of both virulent Pst (EV) and avirulent Pst
(avrRpt2) between PPRL knockout lines and the WT control
(data not shown). It is likely that the possible enhanced resis-
tance was masked by the existing strong resistance to avrRpt2
and, therefore, was difficult to score. However, when PPRL-Flag
overexpression plants were inoculated with a high concentration
(1 ? 107cfu?ml) of Pst (avrRpt2), delayed HR was observed (Fig.
4B) and the transgenic plants displayed considerably less elec-
trolyte leakage at 24 h postinoculation (hpi) (Fig. 4C), which
indicates a reduced level of cell death in the overexpression
plants. Bacterial growth was measured on the plants infected
with a low concentration (2 ? 105cfu?ml) of Pst carrying EV,
avrRpm1, or avrRpt2. The Pst (avrRpt2) titer of the overexpres-
sion line 32 containing a high level of PPRL was ?6- to 8-fold
higher than that of the WT at 4 days postinoculation (dpi). Line
Two lines with high (line 32) or low (line 33) expression level of PPRL were selected for phenotypic analysis. (B) PPRL overexpression line displays delayed HR.
Picture of line 32 was taken at 16 hpi of Pst (avrRpt2) (2 ? 107cfu?ml). (C) PPRL overexpression lines exhibit reduced electrolyte leakage. Plants treated with 10
mM MgCl2and Pst (avrRpt2) (1 ? 107cfu?ml) were measured at 0 and 24 hpi. Error bars represent standard deviation of four replicates. Similar results were
at 0 and 4 dpi of Pst carrying EV, avrRpt2 or avrRpm1 (2 ? 105cfu?ml). Error bars represent standard deviation of five replicates. Similar results were obtained
in three independent experiments. (E) Pst (avrRpt2) accumulates to a higher level in rdr6 and hyl1, but not in dcl3, as compared with that in the corresponding
WT C24, No, or Col-0, respectively. No difference was observed in the growth of Pst (avrRpm1) between the mutants rdr6, hyl1, or dcl3 and their corresponding
WT control. Pathogen growth was measured at 0 and 4 dpi. Similar results were obtained in two independent experiments.
Overexpression of PPRL attenuates RPS2-mediated resistance in Arabidopsis plants. (A) Western blot analysis of transgenic Arabidopsis plants
Katiyar-Agarwal et al.PNAS ?
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33, with a low level of PPRL overexpression, had about a 4- to
5-fold increase in Pst (avrRpt2) bacterial growth than that in the
WT (Fig. 4D). No difference was observed in the growth of Pst
(EV) or Pst (avrRpm1) between PPRL overexpression line and
WT control. These results show that overexpression of PPRL
attenuates RPS2-mediated disease resistance and suggest that
PPRL may function as a negative regulator of the RPS2 pathway.
The induction of nat-siRNAATGB2 is blocked in dcl1-9, hyl1,
and rdr6. Because the dcl1-9 mutation has strong pleiotropic
phenotypes, we chose to examine bacterial growth in rdr6 and
hyl1 mutants. An 8-fold increase of Pst (avrRpt2) bacterial
growth was observed in rdr6 at 4 dpi compared with that in the
WT C24 plants, whereas no significant difference in Pst (avr-
Rpm1) growth was detected (Fig. 4E). Most strikingly, we
observed a complete loss of RPS2-mediated resistance in hyl1,
whereas RPM1-mediated disease resistance was not affected
(Fig. 4E). hyl1 may affect the biogenesis of an array of small
RNAs induced by Pst (avrRpt2), and the elimination of nat-
siRNAATGB2 in hyl1 may contribute a portion of the observed
pathogen susceptibility phenotype. As expected, we did not
detect any obvious difference in pathogen growth between Col-0
WT control and dcl3-1 mutant (Fig. 4E), which affects the
accumulation only of siRNAs associated with chromatin modi-
fication, but not nat-siRNAATGB2 (Fig. 2A). No difference in
pathogen growth was observed between the rdr6, hyl1, or dcl3
mutants and their corresponding WT plants after Pst (avrRpm1)
inoculation (Fig. 4E). Thus, RDR6 and HYL1 play critical roles
in RPS2-mediated resistance pathway by controlling the biogen-
esis of nat-siRNAATGB2 and possibly of other endogenous
Here we identified an endogenous siRNA, nat-siRNAATGB2,
which is specifically induced by Pst (avrRpt2). This nat-siRNA is
produced by a unique biogenesis pathway that requires DCL1,
HYL1, HEN1, RDR6, SGS3, and RNA polymerase IVa (Fig. 5).
Its formation not only requires the induction of the sense
transcript ATGB2, but also depends on the host resistance gene
RPS2 and its resistance signaling components, including NDR1
an intricate regulation of endogenous siRNA formation. The
specific induction of nat-siRNAATGB2 leads to the silencing of
the antisense gene PPRL. Our results suggest that PPRL is a
negative regulator of RPS2 signaling pathway and silencing of
PPRL by nat-siRNAATGB2 plays a positive role in disease
resistance. More than 450 PPR proteins, characterized by the
presence of tandem pentatricopeptide repeats, exist in Arabi-
dopsis and the majority of them have unknown functions (30). A
few studies point to an involvement of PPR proteins in post-
transcriptional processes mainly in organelles, including RNA
editing (31), mRNA silencing by cleavage (32) and translational
regulation (33), etc. The PPRL protein contains five atypical
PPR motifs and is mitochondrial localized (29, 30). Mitochon-
drion is the major organelle involved in oxidative burst and
hypersensitive responses in plant disease resistance and leads to
local cell death. How these events are regulated and how the
signal is transduced are still largely unknown. A recent study
shows that a mitochondrial-localized PPR-containing protein
interacts with inhibitor of apoptosis proteins and regulates
caspase activity and programmed cell death in mammalian cells
(34). We speculate that PPRL may regulate avrRpt2-triggered
oxidative burst, hypersensitive responses, or programmed cell
death, possibly through specific protein–RNA or protein–
protein interactions. Future biochemical analysis on PPRL and
identification of its interaction proteins and RNAs will elucidate
the mechanism of its function in RPS2-mediated bacteria
siRNA-mediated gene silencing plays an essential role in
antiviral defense in both plant and animal systems (35, 36).
However, these siRNAs generated from viral RNAs are ex-
tragenomic in origin. Defense regulation mediated by endoge-
nous small RNAs has been reported in only a few cases thus far,
all of which involve only miRNAs. In mammals, miRNA-
mediated antiviral defense has been reported (37), but the
biological roles of endogenous siRNAs have not been explored.
In plants, miRNA miR393 regulates plant basal defense by
targeting auxin signaling components (8). A direct connection
between endogenous siRNAs and defense responses has not
been reported previously in any organism. Our study here
provides the first example of endogenous siRNAs that play a role
in bacterial disease resistance in Arabidopsis. Gene expression
profiling studies indicate that the defense responses are medi-
ated by activation and repression of a large array of genes, but
how the regulation of gene expression is achieved is largely
unknown. Our data suggest that endogenous siRNA-mediated
gene silencing may serve as one important mechanism for gene
expression reprogramming in plant defense responses. Our
finding of induction of a nat-siRNA in responses to bacterial
infection opens up many new questions and provides new
opportunities to elucidate the molecular events controlling plant
Materials and Methods
Plant Material and Growth Conditions. Arabidopsis thaliana mutants
rdr2-1, dcl2-1, dcl3-1, and dcl4-2, were provided by Jim Carrington
(Center for Genome Research and Biocomputing, Oregon State
University, Corvallis). dcl1-9 and hen1-1 were a gift from Xuemei
Chen. sde1 (rdr6 in this study) and sde4?nrpd1a were provided by
a gift from Herve Vaucheret (Institut National de la Recherche
Agronomique, Versailles, France). hyl1 was a gift from Nina
Federoff (The Huck Institutes of Life Science, Pennsylvania State
Dong (Duke University, Durham, NC). sgt1a and sgt1b were
provided by Jane Parker (Max-Planck-Institut fur Zuchtungsfors-
chung, Cologne, Germany). ein2 was a gift from Athanasios The-
ologis (Plant Gene Expression Center, Albany, CA). eds16 was
jar1 was provided by Linda Walling. These mutants were in the
Columbia (Col-0), Landsberg erecta (Ler), Nossen-0 (No), or C24
plants were grown at 23°C ? 1°C at 12-h light?12-h dark photo-
period. N. benthamiana plants were grown at 23°C ? 1°C at 16-h
light?8-h dark photoperiod.
red are required for nat-siRNAATGB2 formation. RISC, RNA-induced silencing
Model for nat-siRNAATGB2 biogenesis and function. Components in
www.pnas.org?cgi?doi?10.1073?pnas.0608258103 Katiyar-Agarwal et al.
Plasmid DNA Constructs. For generating PPRL overexpression lines, Download full-text
full-length PPRL cDNA without 3? UTR was amplified with
primers 5?-CAC CAT GAA GTT CCT CAT GCA ATC CAT T-3?
the plant expression GATEWAY destination vector p35SGATFH
with C-terminal Flag tag to avoid disruption of the signal peptide
at the N terminus of the protein.
Isolation and Northern Blot Analysis of Small RNAs. Leavesharvested
at 15 hpi of Pst (2 ? 107cfu?ml) were used for RNA extraction and
Northern blot analysis of both high and low molecular weight
in 4 M lithium chloride and precipitated. About 75–120 ?g of
low-molecular-weight RNA was used and separated by 17% dena-
turing polyacrylamide gel. The blots were probed and washed as
Real-time RT-PCR was performed as in ref. 38. PPRL1 was
amplified with primers that locate outside of the overlapping
region: 5?-GCT TCA TCG CCG GAG GAA ATC-3? and
5?-TTA ACC GAG CAC CCT TCA TCG T-3?. Transcript levels
were normalized to that of ubiquitin (5?-CGG AAA GAC CAT
TAC TCT GGA-3? and 5?-CAA GTG TGC GAC CAT CCT
CAA-3?). Each experiment was repeated three times. The
comparative Ct method was applied (ABI User Bulletin No. 2,
Applied Biosystems, West Chester, PA).
Transient Expression Studies in N. benthamiana. A 564-bp overlap-
ping region was amplified with the primers 5?-ACG CGT CGA
CAT GTG GAG CCA CCC GCA GTT CGA AAA ACG TAC
CTA GTG TTA GTG ACG CGA ACA TAC AAT AAC TTG
CG-3?. Full-length ATGB2 was amplified by using primers
5?-ACG CGT CGA CAT GTG GAG CCA CCC GCA GTT
CGA AAA ATC TTA CGA TTA TCT CTT CAA G-3? and
CAA TAA CTT GCG-3?. A strep tag sequence was included in
the forward primers. The amplified products were cloned in
XhoI and SpeI sites of the pTA7002 (28). Agrobacterium tume-
faciens strain GV3101 cells harboring PPRL or ATGB2 con-
structs (OD600? 1.0) were mixed at 1:1 ratio and coinfiltrated
into 3-week-old N. benthamiana leaves. The expression of full-
length or overlapping region of ATGB2 was induced by infiltra-
tion of 30 ?M Dex at 48 hpi, and leaf tissue was collected at 24 h
after Dex induction.
Bacterial Growth Assays. Pst carrying EV (pVSP61) or avrRpt2,
avrRpm1, avrRps4 and avrPphB were used to infect 4-week-old
Arabidopsis leaves by infiltration at a concentration of ?2 ? 105
cfu?ml. The bacterial titer was measured at 0 and 4 dpi as in ref. 38.
HR Assay and Electrolyte Leakage Measurements. Leaves of 4-week-
old Arabidopsis plants were infiltrated with 2 ? 107cfu?ml Pst
(avrRpt2) for HR assay. Leaves were infiltrated with either 10
then transferred into 15 ml of water incubating for 16 h. The
tubes containing leaf disks and water were then autoclaved.
Conductivity was measured before and after autoclave by an EC
meter (VWR Scientific, West Chester, PA). The percentage of
ion leakage before and after autoclave was calculated and
plotted. Four replicates were conducted in each treatment.
We thank Shou-Wei Ding and Xuemei Chen (University of California,
Riverside, CA) and Jim Carrington for stimulating discussion on the
manuscript and for providing seeds of various mutants; David Baul-
combe, Nina Federoff, Herve Vaucheret, Xinnian Dong, Jane Parker,
of California, Riverside) for providing seeds of various genotypes;
Thomas Girke for bioinformatics assistance; Julia Bailey-Serres (Uni-
versity of California, Riverside) for binary plasmids; and James Borne-
man for access to a real-time Icycler in his laboratory. This work was
supported by U.S. Department of Agriculture, State Agricultural Ex-
periment Station Research Allocation Award PPA-7517H from the
University of California, Riverside (to H.J.), Department of Energy
Grant DE-FG02-88ER13917, and National Institutes of Health Grants
R01-FM069680-01 (to B.J.S.) and R01GM59138 and R01GM070795
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vol. 103 ?
no. 47 ?