A plant mechanism of hijacking pathogen virulence factors to trigger innate immunity.

Polygalacturonase-inhibiting proteins (PGIPs) interact with pathogen-derived polygalacturonases to inhibit their virulence-associated plant cell wall-degrading activity but stimulate immunity-inducing oligogalacturonide production. Here we show that interaction between Phaseolus vulgaris PGIP2 (PvPGIP2) and Fusarium phyllophilum polygalacturonase (FpPG) enhances substrate binding, resulting in inhibition of the enzyme activity of FpPG. This interaction promotes FpPG-catalyzed production of long-chain immunoactive oligogalacturonides, while diminishing immunosuppressive short oligogalacturonides. PvPGIP2 binding creates a substrate binding site on PvPGIP2-FpPG, forming a new polygalacturonase with boosted substrate binding activity and altered substrate preference. Structure-based engineering converts a putative PGIP that initially lacks FpPG-binding activity into an effective FpPG-interacting protein. These findings unveil a mechanism for plants to transform pathogen virulence activity into a defense trigger and provide proof of principle for engineering PGIPs with broader specificity.

The NLR immune receptor ADR1 and lipase-like proteins EDS1 and PAD4 mediate stomatal immunity in Nicotiana benthamiana and Arabidopsis.

In the presence of pathogenic bacteria, plants close their stomata to prevent pathogen entry. Intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogenic effectors and activate effector-triggered immune responses. However, the regulatory and molecular mechanisms of stomatal immunity involving NLR immune receptors are unknown. Here, we show that the Nicotiana benthamiana RPW8-NLR central immune receptor ACTIVATED DISEASE RESISTANCE 1 (NbADR1), together with the key immune proteins ENHANCED DISEASE SUSCEPTIBILITY 1 (NbEDS1) and PHYTOALEXIN DEFICIENT 4 (NbPAD4), plays an essential role in bacterial pathogen- and flg22-induced stomatal immunity by regulating the expression of salicylic acid (SA) and abscisic acid (ABA) biosynthesis or response-related genes. NbADR1 recruits NbEDS1 and NbPAD4 in stomata to form a stomatal immune response complex. The transcription factor NbWRKY40e, in association with NbEDS1 and NbPAD4, modulates the expression of SA and ABA biosynthesis or response-related genes to influence stomatal immunity. NbADR1, NbEDS1, and NbPAD4 are required for the pathogen infection-enhanced binding of NbWRKY40e to the ISOCHORISMATE SYNTHASE 1 promoter. Moreover, the ADR1-EDS1-PAD4 module regulates stomatal immunity in Arabidopsis (Arabidopsis thaliana). Collectively, our findings show the pivotal role of the core intracellular immune receptor module ADR1-EDS1-PAD4 in stomatal immunity, which enables plants to limit pathogen entry.

Jasmonate action and crosstalk in flower development and fertility.

Flower development and fertility are coordinately regulated by endogenous developmental signals, including the phytohormones jasmonates (JAs), auxin, and gibberellin, and environmental cues. JAs regulate stamen development and fertility under basal conditions, affect root growth and trichome formation under stress conditions, and control defense responses against insect herbivores and pathogens. Since the 1990s, an increasing number of studies have revealed the essential roles of JA biosynthesis, signaling, and crosstalk in regulation of flower development and fertility. Here, we summarize and present an updated overview of the JA pathway and its crosstalk in modulating flower/sexual organ development and fertility in Arabidopsis, tomato, rice, maize, and sorghum.

Identification of Pathogens and Evaluation of Resistance and Genetic Diversity of Maize Inbred Lines to Stalk Rot in Heilongjiang Province, China.

Maize stalk rot, caused by multiple pathogens, is a serious soilborne disease worldwide. Composition of pathogens causing maize stalk rot and resistance of maize inbred lines in Heilongjiang Province, China, are not well understood. In this study, 138 fungal isolates were collected from different maize-producing areas in Heilongjiang Province, which were identified as Fusarium graminearum (23.2%), F. subglutinans (18.9%), F. cerealis (18.9%), Bipolaris zeicola (13.0%), F. brachygibbosum (13.0%), F. temperatum (7.2%), and F. proliferatum (5.8%). Among them, F. graminearum (>20%) was the predominant species among the isolates causing maize stalk rot. B. zeicola had not previously been reported causing maize stalk rot in China. Resistance of 67 maize inbred lines to maize stalk rot was assessed, and 24 lines (35.8% of them) were highly resistant or resistant, indicating that approximately 65% of these lines were susceptible to maize stalk rot. Maize inbred lines were analyzed using simple sequence repeat markers and divided into five genetic groups with 12 pairs of primers. Additionally, analysis of molecular variance indicated that 44.2% of the genetic variation in disease resistance was distributed among populations. This study provides insight into the genetic diversity of inbred maize and may contribute useful information for breeding stalk rot disease-resistant hybrids, and facilitates development of effective strategies for managing this destructive disease complex.

A molecular framework for signaling crosstalk between jasmonate and ethylene in anthocyanin biosynthesis, trichome development, and defenses against insect herbivores in Arabidopsis.

The phytohormones ethylene (ET) and jasmonate (JA) regulate plant development, growth, and defense responses; however, the molecular basis for their signaling crosstalk is unclear. Here, we show that JA-ZIM-domain (JAZ) proteins, which repress JA signaling, repress trichome initiation/branching and anthocyanin accumulation, and inhibit the transcriptional activity of the basic helix-loop-helix (bHLH)-MYB members (GLABRA3 (GL3)-GL1 and TRANSPARENT TESTA 8 (TT8)-MYB75) of WD-repeat/bHLH/MYB (WBM) complexes. The ET-stabilized transcription factors ETHYLENE-INSENSITIVE3 (EIN3) and EIN3-LIKE1 (EIL1) were found to bind to several members of WBM complexes, including GL3, ENHANCER OF GLABRA3 (EGL3), TT8, GL1, MYB75, and TRANSPARENT TESTA GLABRA1 (TTG1). This binding repressed the transcriptional activity of the bHLH-MYB proteins and inhibited anthocyanin accumulation, trichome formation, and defenses against insect herbivores while promoting root hair formation. Conversely, the JA-activated bHLH members GL3, EGL3, and TT8 of WBM complexes were able to interact with and attenuate the transcriptional activity of EIN3/EIL1 at the HOOKLESS1 promoter, and their overexpression inhibited apical hook formation. Thus, this study demonstrates a molecular framework for signaling crosstalk between JA and ET in plant development, secondary metabolism, and defense responses.

The intragenic suppressor mutation Leu59Phe compensates for the effect of detrimental mutations in the jasmonate receptor COI1.

The phytohormones jasmonates (JAs) control plant development, growth, and defense against insects and pathogens. The Arabidopsis JA receptor Coronatine Insensitive 1 (COI1) interacts with ARABIDOPSIS SKP-LIKE1 (ASK1)/ASK2 to form the SCFCOI1 E3 ligase and mediate JA responses. Here, we performed a genetic suppressor screen using the leaky coi1-2 (COI1Leu245Phe ) mutant for restored sensitivity to JA, and identified the intragenic suppressor mutation Leu59Phe, which was in the region connecting the F-box and leucine-rich repeats domains of COI1. The L59F substitution not only restores the COI1L245F function, but also the COI1Gly434Glu (coi1-22rsp ) function in JA responses, through recovering their interactions with ASK1 or ASK2 and their protein levels. The L59F change itself could not enhance the interactions between COI1 and ASK1/2, nor affect JA responses. The present study reveals that the Leu59Phe substitution compensates for the effect of some deleterious mutations in the JA receptor COI1.

Loss of function of a DMR6 ortholog in tomato confers broad-spectrum disease resistance.

Plant diseases are among the major causes of crop yield losses around the world. To confer disease resistance, conventional breeding relies on the deployment of single resistance (R) genes. However, this strategy has been easily overcome by constantly evolving pathogens. Disabling susceptibility (S) genes is a promising alternative to R genes in breeding programs, as it usually offers durable and broad-spectrum disease resistance. In Arabidopsis, the S gene DMR6 (AtDMR6) encodes an enzyme identified as a susceptibility factor to bacterial and oomycete pathogens. Here, we present a model-to-crop translational work in which we characterize two AtDMR6 orthologs in tomato, SlDMR6-1 and SlDMR6-2. We show that SlDMR6-1, but not SlDMR6-2, is up-regulated by pathogen infection. In agreement, Sldmr6-1 mutants display enhanced resistance against different classes of pathogens, such as bacteria, oomycete, and fungi. Notably, disease resistance correlates with increased salicylic acid (SA) levels and transcriptional activation of immune responses. Furthermore, we demonstrate that SlDMR6-1 and SlDMR6-2 display SA-5 hydroxylase activity, thus contributing to the elucidation of the enzymatic function of DMR6. We then propose that SlDMR6 duplication in tomato resulted in subsequent subfunctionalization, in which SlDMR6-2 specialized in balancing SA levels in flowers/fruits, while SlDMR6-1 conserved the ability to fine-tune SA levels during pathogen infection of the plant vegetative tissues. Overall, this work not only corroborates a mechanism underlying SA homeostasis in plants, but also presents a promising strategy for engineering broad-spectrum and durable disease resistance in crops.

Functional specificity, diversity, and redundancy of Arabidopsis JAZ family repressors in jasmonate and COI1-regulated growth, development, and defense.

In response to jasmonates (JAs), the JA receptor Coronatine Insensitive 1 (COI1) recruits JA-zinc-finger inflorescence meristem (ZIM)-domain (JAZ) family repressors for destruction to regulate plant growth, development, and defense. As Arabidopsis encodes 13 JAZ repressors, their functional specificity, diversity, and redundancy in JA/COI1-mediated responses remain unclear. We generated a broad range of jaz mutants based on their phylogenetic relationship to investigate their roles in JA responses. The group I JAZ6 may play an inhibitory role in resistance to Botrytis cinerea, group II (JAZ10)/III (JAZ11/12) in JA-regulated root growth inhibition and susceptibility to Pseudomonas syringae pv tomato DC3000, and group IV JAZ3/4/9 in flowering time delay and defense against insects. JAZs exhibit high redundancy in apical hook curvature. The undecuple jaz1/2/3/4/5/6/7/9/10/11/12 (jaz1-7,9-12) mutations enhance JA responses and suppress the phenotypes of coi1-1 in flowering time, rosette growth, and defense. The JA hypersensitivity of jaz1-7,9-12 in root growth, hook curvature, and leaf yellowing is blocked by coi1-1. jaz1-7,9-12 does not influence the stamen phenotypes of wild-type and coi1-1. jaz1-7,9-12 affects JA-regulated transcriptional profile and recovers a fraction of that in coi1-1. This study contributes to elucidating the specificity, diversity, and redundancy of JAZ members in JA/COI1-regulated growth, development, and defense responses.

Structure of the activated ROQ1 resistosome directly recognizing the pathogen effector XopQ.

Plants and animals detect pathogen infection using intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) that directly or indirectly recognize pathogen effectors and activate an immune response. How effector sensing triggers NLR activation remains poorly understood. Here we describe the 3.8-angstrom-resolution cryo-electron microscopy structure of the activated ROQ1 (recognition of XopQ 1), an NLR native to Nicotiana benthamiana with a Toll-like interleukin-1 receptor (TIR) domain bound to the Xanthomonas euvesicatoria effector XopQ (Xanthomonas outer protein Q). ROQ1 directly binds to both the predicted active site and surface residues of XopQ while forming a tetrameric resistosome that brings together the TIR domains for downstream immune signaling. Our results suggest a mechanism for the direct recognition of effectors by NLRs leading to the oligomerization-dependent activation of a plant resistosome and signaling by the TIR domain.

NAD+ cleavage activity by animal and plant TIR domains in cell death pathways.

SARM1 (sterile alpha and TIR motif containing 1) is responsible for depletion of nicotinamide adenine dinucleotide in its oxidized form (NAD+) during Wallerian degeneration associated with neuropathies. Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effector proteins and trigger localized cell death to restrict pathogen infection. Both processes depend on closely related Toll/interleukin-1 receptor (TIR) domains in these proteins, which, as we show, feature self-association-dependent NAD+ cleavage activity associated with cell death signaling. We further show that SARM1 SAM (sterile alpha motif) domains form an octamer essential for axon degeneration that contributes to TIR domain enzymatic activity. The crystal structures of ribose and NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) complexes of SARM1 and plant NLR RUN1 TIR domains, respectively, reveal a conserved substrate binding site. NAD+ cleavage by TIR domains is therefore a conserved feature of animal and plant cell death signaling pathways.

Using forward genetics in Nicotiana benthamiana to uncover the immune signaling pathway mediating recognition of the Xanthomonas perforans effector XopJ4.

The immune pathway responsible for perception of the Xanthomonas perforans effector XopJ4 was identified in the plant Nicotiana benthamiana. This pathogen causes significant yield loss in commercial tomato cultivation. Genetic mapping and viral-induced gene silencing were used to identify immune signaling components of the XopJ4 perception pathway in N. benthamiana. Transient complementation assays were performed to determine the functionality of gene variants and co-immunoprecipitation assays were used to gain insight into the molecular mechanism of the pathway. Two N. benthamiana ethyl methanesulfonate (EMS) mutants deficient for XopJ4 perception were identified as having loss-of-function mutations in the gene encoding the nucleotide binding, leucine-rich repeat (NLR) protein NbZAR1. Silencing of a receptor-like cytoplasmic kinase family XII gene, subsequently named XOPJ4 IMMUNITY 2 (JIM2), blocks perception of XopJ4. This study demonstrates the feasibility of conducting mutant screens in N. benthamiana to investigate the genetic basis of the plant immune system and other processes. The identification of NbZAR1 and JIM2 as mediating XopJ4 perception in N. benthamiana supports the model of ZAR1 being involved in the perception of many different pathogen effector proteins with specificity dictated by associated receptor-like cytoplasmic kinases.

NRG1 functions downstream of EDS1 to regulate TIR-NLR-mediated plant immunity in Nicotiana benthamiana.

Effector-triggered immunity (ETI) in plants involves a large family of nucleotide-binding leucine-rich repeat (NLR) immune receptors, including Toll/IL-1 receptor-NLRs (TNLs) and coiled-coil NLRs (CNLs). Although various NLR immune receptors are known, a mechanistic understanding of NLR function in ETI remains unclear. The TNL Recognition of XopQ 1 (Roq1) recognizes the effectors XopQ and HopQ1 from Xanthomonas and Pseudomonas, respectively, which activates resistance to Xanthomonas euvesicatoria and Xanthomonas gardneri in an Enhanced Disease Susceptibility 1 (EDS1)-dependent way in Nicotiana benthamiana In this study, we found that the N. benthamiana N requirement gene 1 (NRG1), a CNL protein required for the tobacco TNL protein N-mediated resistance to tobacco mosaic virus, is also essential for immune signaling [including hypersensitive response (HR)] triggered by the TNLs Roq1 and Recognition of Peronospora parasitica 1 (RPP1), but not by the CNLs Bs2 and Rps2, suggesting that NRG1 may be a conserved key component in TNL signaling pathways. Besides EDS1, Roq1 and NRG1 are necessary for resistance to Xanthomonas and Pseudomonas in N. benthamiana NRG1 functions downstream of Roq1 and EDS1 and physically associates with EDS1 in mediating XopQ-Roq1-triggered immunity. Moreover, RNA sequencing analysis showed that XopQ-triggered gene-expression profile changes in N. benthamiana were almost entirely mediated by Roq1 and EDS1 and were largely regulated by NRG1. Overall, our study demonstrates that NRG1 is a key component that acts downstream of EDS1 to mediate various TNL signaling pathways, including Roq1 and RPP1-mediated HR, resistance to Xanthomonas and Pseudomonas, and XopQ-regulated transcriptional changes in N. benthamiana.

bHLH13 Regulates Jasmonate-Mediated Defense Responses and Growth.

Jasmonates (JAs) regulate plant growth and defense responses. On perception of bioactive JAs, the JA receptor CORONATINE INSENSITIVE1 (COI1) recruits JA ZIM-domain (JAZ) proteins for degradation, and JAZ-targeted transcription factors are released to regulate JA responses. The subgroup IIId bHLH transcriptional factors, including bHLH17, bHLH13, bHLH3, and bHLH14, interact with JAZs and repress JA responses. In this study, we show that IIId bHLH factors form dimers via the C-terminus in yeast. N-terminus of bHLH13 is essential for its transcriptional repression function. bHLH13 overexpression inhibits Arabidopsis resistance to the necrotrophic fungi Botrytis cinerea and defense against the insect Spodoptera exigua. COI1 mutation disrupts the oversensitivity of the quadruple mutant bhlh3 bhlh13 bhlh14 bhlh17 in various JA responses, including anthocyanin accumulation, root growth inhibition, and defense against B cinerea and S exigua. Disruption of the TTG1/bHLH/MYB complex blocks anthocyanin accumulation of bhlh3 bhlh13 bhlh14 bhlh17, whereas abolishment of MYC2 attenuates JA-inhibitory root growth of bhlh3 bhlh13 bhlh14 bhlh17. These results genetically demonstrate that IIId bHLH factors function downstream of COI1 to inhibit distinctive JA responses via antagonizing different transcriptional activators.

Arabidopsis ENOR3 regulates RNAi-mediated antiviral defense.

Viruses can infect host plants to cause severe diseases and substantial agricultural loss, while plants have evolved RNA interference (RNAi) strategy to defend against viral infection. Despite enormous efforts, only a few host proteins in RNAi pathway were shown to mediate antiviral defense, including RNA-dependent RNA polymerase 1 (RDR1), RDR6, DICER-LIKE 2 (DCL2) and DCL4. In this study, we carried out a genetic screen for antiviral factors of RNAi pathway in Arabidopsis rdr6 background via inoculation with a 2b-deficient Cucumber Mosaic Virus (CMV-Δ2b). We identified a mutant susceptible to CMV-Δ2b, referred to as enhancer of rdr6 (enor) 3-1 rdr6, and found that ENOR3 encodes a functionally unknown protein with high homology to the mammalian Non Imprinted in Prader-Willi/Angelman (NIPA) magnesium transporters. ENOR3 inhibits accumulation of CMV-Δ2b and acts additively with RDR1, RDR6, DCL2 and DCL4 in antiviral defense. These results uncover that ENOR3 is a key component in antiviral RNAi pathway, and provide new insights into antiviral immunity.

The C-terminal domains of Arabidopsis GL3/EGL3/TT8 interact with JAZ proteins and mediate dimeric interactions.

The phytohormone jasmonates (JAs) regulate plant development, growth, secondary metabolism, and defense responses. JAs act through CORONATINE INSENSITIVE1 (COI1) to induce the degradation of JA ZIM-domain (JAZ) proteins, and activate JAZ-repressed transcription factors to regulate plant response. We previously showed that the basic helix-loop-helix (bHLH) and MYB members of the WD-repeat/bHLH/MYB complex interacted with JAZs and mediated JA-induced anthocyanin accumulation and trichome initiation. In this study, we showed that the C-terminal domain of the bHLH members (GLABRA3 [GL3], ENHANCER OF GLABRA3 [EGL3] and TRANSPARENT TESTA8 [TT8]) interacted with JAZs in yeast and plant, and mediated dimerizations between the bHLH members. Our study provides further understanding of the bHLH members of the WD-repeat/bHLH/MYB complex in JA pathway.

MYC5 is Involved in Jasmonate-Regulated Plant Growth, Leaf Senescence and Defense Responses.

Jasmonates (JAs), lipid-derived phytohormones, regulate plant growth, development and defenses against biotic stresses. CORONATINE INSENSITIVE1 perceives bioactive JA and recruits JASMONATE ZIM-DOMAIN (JAZ) proteins for ubiquitination and subsequent degradation via the 26S proteasome, which de-represses JAZ-targeted transcription factors that regulate diverse JA responses. Recent studies showed that the Arabidopsis basic helix-loop-helix transcription factor MYC5 interacts with JAZs and regulates stamen development. However, whether MYC5 mediates other JA responses is unclear. Here, we show that MYC5 functions redundantly with MYC2, MYC3 and MYC4 to modulate JA-regulated root growth inhibition and plant defenses against insect attack and pathogen infection, and that it positively regulates JA-induced leaf senescence. Our findings define MYC5 as an important regulator that is essential for diverse JA responses.

Arabidopsis MYB24 Regulates Jasmonate-Mediated Stamen Development.

The phytohormone jasmonates (JAs) regulate various defense responses and diverse developmental processes including stamen development and fertility. Previous studies showed that JA induces CORONATINE INSENSITIVE 1-mediated degradation of JA ZIM-domain (JAZ) proteins, and activates the MYB transcription factors (such as MYB21 and MYB24) to regulate stamen development. In this study, we further uncover the mechanism underlying how MYB24 interacts with JAZs to control JA-regulated stamen development. We show that N-terminus of MYB21/24 interacts with 10 out of 12 JAZ proteins while both N-terminus and C-terminus of MYB24 are involved in dimerization of MYB21 and MYB24. Interestingly, male sterility of the JA-deficient mutant opr3 can be rescued by suitable level of the MYB24 overexpression but not by excessive high level of MYB24. Surprisingly, overexpression of MYB24NT, but not MYB24CT, could cause male sterility. These results provide new insights on MYB factors in JA-regulated stamen development.

Roq1 mediates recognition of the Xanthomonas and Pseudomonas effector proteins XopQ and HopQ1.

Xanthomonas spp. are phytopathogenic bacteria that can cause disease on a wide variety of plant species resulting in significant impacts on crop yields. Limited genetic resistance is available in most crop species and current control methods are often inadequate, particularly when environmental conditions favor disease. The plant Nicotiana benthamiana has been shown to be resistant to Xanthomonas and Pseudomonas due to an immune response triggered by the bacterial effector proteins XopQ and HopQ1, respectively. We used a reverse genetic screen to identify Recognition of XopQ 1 (Roq1), a nucleotide-binding leucine-rich repeat (NLR) protein with a Toll-like interleukin-1 receptor (TIR) domain, which mediates XopQ recognition in N. benthamiana. Roq1 orthologs appear to be present only in the Nicotiana genus. Expression of Roq1 was found to be sufficient for XopQ recognition in both the closely-related Nicotiana sylvestris and the distantly-related beet plant (Beta vulgaris). Roq1 was found to co-immunoprecipitate with XopQ, suggesting a physical association between the two proteins. Roq1 is able to recognize XopQ alleles from various Xanthomonas species, as well as HopQ1 from Pseudomonas, demonstrating widespread potential application in protecting crop plants from these pathogens.

GDP-D-mannose epimerase regulates male gametophyte development, plant growth and leaf senescence in Arabidopsis.

Plant GDP-D-mannose epimerase (GME) converts GDP-D-mannose to GDP-L-galactose, a precursor of both L-ascorbate (vitamin C) and cell wall polysaccharides. However, the genetic functions of GME in Arabidopsis are unclear. In this study, we found that mutations in Arabidopsis GME affect pollen germination, pollen tube elongation, and transmission and development of the male gametophyte through analysis of the heterozygous GME/gme plants and the homozygous gme plants. Arabidopsis gme mutants also exhibit severe growth defects and early leaf senescence. Surprisingly, the defects in male gametophyte in the gme plants are not restored by L-ascorbate, boric acid or GDP-L-galactose, though boric acid rescues the growth defects of the mutants, indicating that GME may regulate male gametophyte development independent of L-ascorbate and GDP-L-galactose. These results reveal key roles for Arabidopsis GME in reproductive development, vegetative growth and leaf senescence, and suggest that GME regulates plant growth and controls male gametophyte development in different manners.

Arabidopsis ALA1 and ALA2 Mediate RNAi-Based Antiviral Immunity.

RNA intereferencing (RNAi) pathway regulates antiviral immunity and mediates plant growth and development. Despite considerable research efforts, a few components in RNAi pathway have been revealed, including ARGONAUTEs (AGOs), DICER-LIKEs (DCLs), RNA-dependent RNA polymerase 1 and 6 (RDR1/6), and ALTERED MERISTEM PROGRAM 1 (AMP1). In this study, we performed a forward genetic screening for enhancers of rdr6 via inoculation of CMV2aTΔ2b, a 2b-deficient Cucumber Mosaic Virus that is unable to suppress RNAi-mediated antiviral immunity. We uncover that the membrane-localized flippase Aminophospholipid ATPase 1 (ALA1) cooperates with RDR6 and RDR1 to promote antiviral immunity and regulate fertility in Arabidopsis. Moreover, we find that ALA2, a homolog of ALA1, also participates in antiviral immunity. Our findings suggest that ALA1 and ALA2 act as novel components in the RNAi pathway and function additively with RDR1 and RDR6 to mediate RNAi-based antiviral immunity and plant development.