Image-Based Quantification of Arabidopsis thaliana Stomatal Aperture from Leaf Images.

The quantification of stomatal pore size has long been a fundamental approach to understand the physiological response of plants in the context of environmental adaptation. Automation of such methodologies not only alleviates human labor and bias but also realizes new experimental research methods through massive analysis. Here, we present an image analysis pipeline that automatically quantifies stomatal aperture of Arabidopsis thaliana leaves from bright-field microscopy images containing mesophyll tissue as noisy backgrounds. By combining a You Only Look Once X-based stomatal detection submodule and a U-Net-based pore segmentation submodule, we achieved a mean average precision with an intersection of union (IoU) threshold of 50% value of 0.875 (stomata detection performance) and an IoU of 0.745 (pore segmentation performance) against images of leaf discs taken with a bright-field microscope. Moreover, we designed a portable imaging device that allows easy acquisition of stomatal images from detached/undetached intact leaves on-site. We demonstrated that this device in combination with fine-tuned models of the pipeline we generated here provides robust measurements that can substitute for manual measurement of stomatal responses against pathogen inoculation. Utilization of our hardware and pipeline for automated stomatal aperture measurements is expected to accelerate research on stomatal biology of model dicots.

The rice OsERF101 transcription factor regulates the NLR Xa1-mediated immunity induced by perception of TAL effectors.

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) initiate immune responses by recognizing pathogen effectors. The rice gene Xa1 encodes an NLR with an N-terminal BED domain, and recognizes transcription activator-like (TAL) effectors of Xanthomonas oryzae pv oryzae (Xoo). Our goal here was to elucidate the molecular mechanisms controlling the induction of immunity by Xa1. We used yeast two-hybrid assays to screen for host factors that interact with Xa1 and identified the AP2/ERF-type transcription factor OsERF101/OsRAP2.6. Molecular complementation assays were used to confirm the interactions among Xa1, OsERF101 and two TAL effectors. We created OsERF101-overexpressing and knockout mutant lines in rice and identified genes differentially regulated in these lines, many of which are predicted to be involved in the regulation of response to stimulus. Xa1 interacts in the nucleus with the TAL effectors and OsERF101 via the BED domain. Unexpectedly, both the overexpression and the knockout lines of OsERF101 displayed Xa1-dependent, enhanced resistance to an incompatible Xoo strain. Different sets of genes were up- or downregulated in the overexpression and knockout lines. Our results indicate that OsERF101 regulates the recognition of TAL effectors by Xa1, and functions as a positive regulator of Xa1-mediated immunity. Furthermore, an additional Xa1-mediated immune pathway is negatively regulated by OsERF101.

Overexpression of NDR1 leads to pathogen resistance at elevated temperatures.

Abiotic and biotic environments influence a myriad of plant-related processes, including growth, development, and the establishment and maintenance of interaction(s) with microbes. In the case of the latter, elevated temperature has been shown to be a key factor that underpins host resistance and pathogen virulence. In this study, we elucidate a role for Arabidopsis NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1) by exploiting effector-triggered immunity to define the regulation of plant host immunity in response to both pathogen infection and elevated temperature. We generated time-series RNA sequencing data of WT Col-0, an NDR1 overexpression line, and ndr1 and ics1-2 mutant plants under elevated temperature. Not surprisingly, the NDR1-overexpression line showed genotype-specific gene expression changes related to defense response and immune system function. The results described herein support a role for NDR1 in maintaining cell signaling during simultaneous exposure to elevated temperature and avirulent pathogen stressors.

Balancing trade-offs between biotic and abiotic stress responses through leaf age-dependent variation in stress hormone cross-talk.

In nature, plants must respond to multiple stresses simultaneously, which likely demands cross-talk between stress-response pathways to minimize fitness costs. Here we provide genetic evidence that biotic and abiotic stress responses are differentially prioritized in Arabidopsis thaliana leaves of different ages to maintain growth and reproduction under combined biotic and abiotic stresses. Abiotic stresses, such as high salinity and drought, blunted immune responses in older rosette leaves through the phytohormone abscisic acid signaling, whereas this antagonistic effect was blocked in younger rosette leaves by PBS3, a signaling component of the defense phytohormone salicylic acid. Plants lacking PBS3 exhibited enhanced abiotic stress tolerance at the cost of decreased fitness under combined biotic and abiotic stresses. Together with this role, PBS3 is also indispensable for the establishment of salt stress- and leaf age-dependent phyllosphere bacterial communities. Collectively, our work reveals a mechanism that balances trade-offs upon conflicting stresses at the organism level and identifies a genetic intersection among plant immunity, leaf microbiota, and abiotic stress tolerance.

The Defense Phytohormone Signaling Network Enables Rapid, High-Amplitude Transcriptional Reprogramming during Effector-Triggered Immunity.

The phytohormone network consisting of jasmonate, ethylene, PHYTOALEXIN-DEFICIENT4, and salicylic acid signaling is required for the two modes of plant immunity, pattern-triggered immunity (PTI), and effector-triggered immunity (ETI). A previous study showed that during PTI, the transcriptional responses of over 5000 genes qualitatively depend on complex interactions between the network components. However, the role of the network in transcriptional reprogramming during ETI and whether it differs between PTI and ETI remain elusive. Here, we generated time-series RNA-sequencing data of Arabidopsis thaliana wild-type and combinatorial mutant plants deficient in components of the network upon challenge with virulent or ETI-triggering avirulent strains of the foliar bacterial pathogen Pseudomonas syringae Resistant plants such as the wild type achieved high-amplitude transcriptional reprogramming 4 h after challenge with avirulent strains and sustained this transcriptome response. Strikingly, susceptible plants including the quadruple network mutant showed almost identical transcriptome responses to resistant plants but with several hours delay. Furthermore, gene coexpression network structure was highly conserved between the wild type and quadruple mutant. Thus, in contrast to PTI, the phytohormone network is required only for achieving high-amplitude transcriptional reprogramming within the early time window of ETI against this bacterial pathogen.

The Arabidopsis CERK1-associated kinase PBL27 connects chitin perception to MAPK activation.

Perception of microbe-associated molecular patterns by host cell surface pattern recognition receptors (PRRs) triggers the intracellular activation of mitogen-activated protein kinase (MAPK) cascades. However, it is not known how PRRs transmit immune signals to MAPK cascades in plants. Here, we identify a complete phospho-signaling transduction pathway from PRR-mediated pathogen recognition to MAPK activation in plants. We found that the receptor-like cytoplasmic kinase PBL27 connects the chitin receptor complex CERK1-LYK5 and a MAPK cascade. PBL27 interacts with both CERK1 and the MAPK kinase kinase MAPKKK5 at the plasma membrane. Knockout mutants of MAPKKK5 compromise chitin-induced MAPK activation and disease resistance to Alternaria brassicicola PBL27 phosphorylates MAPKKK5 in vitro, which is enhanced by phosphorylation of PBL27 by CERK1. The chitin perception induces disassociation between PBL27 and MAPKKK5 in vivo Furthermore, genetic evidence suggests that phosphorylation of MAPKKK5 by PBL27 is essential for chitin-induced MAPK activation in plants. These data indicate that PBL27 is the MAPKKK kinase that provides the missing link between the cell surface chitin receptor and the intracellular MAPK cascade in plants.

Pathogen exploitation of an abscisic acid- and jasmonate-inducible MAPK phosphatase and its interception by Arabidopsis immunity.

Phytopathogens promote virulence by, for example, exploiting signaling pathways mediated by phytohormones such as abscisic acid (ABA) and jasmonate (JA). Some plants can counteract pathogen virulence by invoking a potent form of immunity called effector-triggered immunity (ETI). Here, we report that ABA and JA mediate inactivation of the immune-associated MAP kinases (MAPKs), MPK3 and MPK6, in Arabidopsis thaliana ABA induced expression of genes encoding the protein phosphatases 2C (PP2Cs), HAI1, HAI2, and HAI3 through ABF/AREB transcription factors. These three HAI PP2Cs interacted with MPK3 and MPK6 and were required for ABA-mediated MPK3/MPK6 inactivation and immune suppression. The bacterial pathogen Pseudomonas syringae pv. tomato (Pto) DC3000 activates ABA signaling and produces a JA-mimicking phytotoxin, coronatine (COR), that promotes virulence. We found that Pto DC3000 induces HAI1 through COR-mediated activation of MYC2, a master transcription factor in JA signaling. HAI1 dephosphorylated MPK3 and MPK6 in vitro and was necessary for COR-mediated suppression of MPK3/MPK6 activation and immunity. Intriguingly, upon ETI activation, A. thaliana plants overcame the HAI1-dependent virulence of COR by blocking JA signaling. Finally, we showed conservation of induction of HAI PP2Cs by ABA and JA in other Brassicaceae species. Taken together, these results suggest that ABA and JA signaling pathways, which are hijacked by the bacterial pathogen, converge on the HAI PP2Cs that suppress activation of the immune-associated MAPKs. Also, our data unveil interception of JA-signaling activation as a host counterstrategy against the bacterial suppression of MAPKs during ETI.

An incoherent feed-forward loop mediates robustness and tunability in a plant immune network.

Immune signaling networks must be tunable to alleviate fitness costs associated with immunity and, at the same time, robust against pathogen interferences. How these properties mechanistically emerge in plant immune signaling networks is poorly understood. Here, we discovered a molecular mechanism by which the model plant species Arabidopsis thaliana achieves robust and tunable immunity triggered by the microbe-associated molecular pattern, flg22. Salicylic acid (SA) is a major plant immune signal molecule. Another signal molecule jasmonate (JA) induced expression of a gene essential for SA accumulation, EDS5 Paradoxically, JA inhibited expression of PAD4, a positive regulator of EDS5 expression. This incoherent type-4 feed-forward loop (I4-FFL) enabled JA to mitigate SA accumulation in the intact network but to support it under perturbation of PAD4, thereby minimizing the negative impact of SA on fitness as well as conferring robust SA-mediated immunity. We also present evidence for evolutionary conservation of these gene regulations in the family Brassicaceae Our results highlight an I4-FFL that simultaneously provides the immune network with robustness and tunability in A. thaliana and possibly in its relatives.

A dominant-interfering camta3 mutation compromises primary transcriptional outputs mediated by both cell surface and intracellular immune receptors in Arabidopsis thaliana.

Pattern recognition receptors (PRRs) and nucleotide-binding domain and leucine-rich repeat (LRR)-containing proteins (NLRs) initiate pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), respectively, each associated with the activation of an overlapping set of defence genes. The regulatory mechanism behind this convergence of PTI- and ETI-mediated defence gene induction remains elusive. We generated transgenic Arabidopsis plants that enable conditional NLR activation without pathogen infection to dissect NLR- and PRR-mediated transcriptional signals. A comparative analysis of over 40 transcriptome datasets linked calmodulin-binding transcription activators (CAMTAs) to the activation of overlapping defence genes in PTI and ETI. We used a dominant camta3 mutant (camta3-D) to assess CAMTA functions in the corresponding transcriptional regulation. Transcriptional regulation by NLRs, although highly similar to PTI responses, can be established independently of pathogen-associated molecular pattern (PAMP) perception, defence phytohormones and host cell death. Conditional expression of the N-terminal coiled-coil domain of the barley MLA (Mildew resistance locus A) NLR is sufficient to trigger similar transcriptional reprogramming as full-length NLRs. CAMTA-binding motifs are overrepresented in the 5' regulatory regions of the identified primary immune response genes, consistent with their altered expression and disease resistance responses in camta3-D plants. We propose that CAMTA-mediated transcriptional regulation defines an early convergence point in NLR- and PRR-mediated signalling.

GAPDH--a recruits a plant virus movement protein to cortical virus replication complexes to facilitate viral cell-to-cell movement.

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process.

Dual regulation of gene expression mediated by extended MAPK activation and salicylic acid contributes to robust innate immunity in Arabidopsis thaliana.

Network robustness is a crucial property of the plant immune signaling network because pathogens are under a strong selection pressure to perturb plant network components to dampen plant immune responses. Nevertheless, modulation of network robustness is an area of network biology that has rarely been explored. While two modes of plant immunity, Effector-Triggered Immunity (ETI) and Pattern-Triggered Immunity (PTI), extensively share signaling machinery, the network output is much more robust against perturbations during ETI than PTI, suggesting modulation of network robustness. Here, we report a molecular mechanism underlying the modulation of the network robustness in Arabidopsis thaliana. The salicylic acid (SA) signaling sector regulates a major portion of the plant immune response and is important in immunity against biotrophic and hemibiotrophic pathogens. In Arabidopsis, SA signaling was required for the proper regulation of the vast majority of SA-responsive genes during PTI. However, during ETI, regulation of most SA-responsive genes, including the canonical SA marker gene PR1, could be controlled by SA-independent mechanisms as well as by SA. The activation of the two immune-related MAPKs, MPK3 and MPK6, persisted for several hours during ETI but less than one hour during PTI. Sustained MAPK activation was sufficient to confer SA-independent regulation of most SA-responsive genes. Furthermore, the MPK3 and SA signaling sectors were compensatory to each other for inhibition of bacterial growth as well as for PR1 expression during ETI. These results indicate that the duration of the MAPK activation is a critical determinant for modulation of robustness of the immune signaling network. Our findings with the plant immune signaling network imply that the robustness level of a biological network can be modulated by the activities of network components.

ADP ribosylation factor 1 plays an essential role in the replication of a plant RNA virus.

Eukaryotic positive-strand RNA viruses replicate using the membrane-bound replicase complexes, which contain multiple viral and host components. Virus infection induces the remodeling of intracellular membranes. Virus-induced membrane structures are thought to increase the local concentration of the components that are required for replication and provide a scaffold for tethering the replicase complexes. However, the mechanisms underlying virus-induced membrane remodeling are poorly understood. RNA replication of red clover necrotic mosaic virus (RCNMV), a positive-strand RNA plant virus, is associated with the endoplasmic reticulum (ER) membranes, and ER morphology is perturbed in RCNMV-infected cells. Here, we identified ADP ribosylation factor 1 (Arf1) in the affinity-purified RCNMV RNA-dependent RNA polymerase fraction. Arf1 is a highly conserved, ubiquitous, small GTPase that is implicated in the formation of the coat protein complex I (COPI) vesicles on Golgi membranes. Using in vitro pulldown and bimolecular fluorescence complementation analyses, we showed that Arf1 interacted with the viral p27 replication protein within the virus-induced large punctate structures of the ER membrane. We found that inhibition of the nucleotide exchange activity of Arf1 using the inhibitor brefeldin A (BFA) disrupted the assembly of the viral replicase complex and p27-mediated ER remodeling. We also showed that BFA treatment and the expression of dominant negative Arf1 mutants compromised RCNMV RNA replication in protoplasts. Interestingly, the expression of a dominant negative mutant of Sar1, a key regulator of the biogenesis of COPII vesicles at ER exit sites, also compromised RCNMV RNA replication. These results suggest that the replication of RCNMV depends on the host membrane traffic machinery.

Sugar coordinates plant defense signaling.

Pathogen recognition triggers energy-intensive defense systems. Although successful defense should depend on energy availability, how metabolic information is communicated to defense remains unclear. We show that sugar, especially glucose-6-phosphate (G6P), is critical in coordinating defense in Arabidopsis. Under sugar-sufficient conditions, phosphorylation levels of calcium-dependent protein kinase 5 (CPK5) are elevated by G6P-mediated suppression of protein phosphatases, enhancing defense responses before pathogen invasion. Subsequently, recognition of bacterial flagellin activates sugar transporters, leading to increased cellular G6P, which elicits CPK5-independent signaling promoting synthesis of the phytohormone salicylic acid (SA) for antibacterial defense. In contrast, while perception of fungal chitin does not promote sugar influx or SA accumulation, chitin-induced synthesis of the antifungal compound camalexin requires basal sugar influx activity. By monitoring sugar levels, plants determine defense levels and execute appropriate outputs against bacterial and fungal pathogens. Together, our findings provide a comprehensive view of the roles of sugar in defense.

Direct Observation and Automated Measurement of Stomatal Responses to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana.

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake for photosynthesis and transpirational water loss but also for restricting bacterial invasion. While plants close stomata upon recognition of microbes, pathogenic bacteria, such as Pseudomonas syringae pv. tomato DC3000 (Pto), reopen the closed stomata to gain access into the leaf interior. In conventional assays for assessing stomatal responses to bacterial invasion, leaf epidermal peels, leaf discs, or detached leaves are floated on bacterial suspension, and then stomata are observed under a microscope followed by manual measurement of stomatal aperture. However, these assays are cumbersome and may not reflect stomatal responses to natural bacterial invasion in a leaf attached to the plant. Recently, a portable imaging device was developed that can observe stomata by pinching a leaf without detaching it from the plant, together with a deep learning-based image analysis pipeline designed to automatically measure stomatal aperture from leaf images captured by the device. Here, building on these technical advances, a new method to assess stomatal responses to bacterial invasion in Arabidopsis thaliana is introduced. This method consists of three simple steps: spray inoculation of Pto mimicking natural infection processes, direct observation of stomata on a leaf of the Pto-inoculated plant using the portable imaging device, and automated measurement of stomatal aperture by the image analysis pipeline. This method was successfully used to demonstrate stomatal closure and reopening during Pto invasion under conditions that closely mimic the natural plant-bacteria interaction.

Commensal lifestyle regulated by a negative feedback loop between Arabidopsis ROS and the bacterial T2SS.

Despite the plant health-promoting effects of plant microbiota, these assemblages also comprise potentially detrimental microbes. How plant immunity controls its microbiota to promote plant health under these conditions remains largely unknown. We find that commensal bacteria isolated from healthy Arabidopsis plants trigger diverse patterns of reactive oxygen species (ROS) production dependent on the immune receptors and completely on the NADPH oxidase RBOHD that selectively inhibited specific commensals, notably Xanthomonas L148. Through random mutagenesis, we find that L148 gspE, encoding a type II secretion system (T2SS) component, is required for the damaging effects of Xanthomonas L148 on rbohD mutant plants. In planta bacterial transcriptomics reveals that RBOHD suppresses most T2SS gene expression including gspE. L148 colonization protected plants against a bacterial pathogen, when gspE was inhibited by ROS or mutation. Thus, a negative feedback loop between Arabidopsis ROS and the bacterial T2SS tames a potentially detrimental leaf commensal and turns it into a microbe beneficial to the host.

Differential roles of Hsp70 and Hsp90 in the assembly of the replicase complex of a positive-strand RNA plant virus.

Assembly of viral replicase complexes of eukaryotic positive-strand RNA viruses is a regulated process: multiple viral and host components must be assembled on intracellular membranes and ordered into quaternary complexes capable of synthesizing viral RNAs. However, the molecular mechanisms underlying this process are poorly understood. In this study, we used a model virus, Red clover necrotic mosaic virus (RCNMV), whose replicase complex can be detected readily as the 480-kDa functional protein complex. We found that host heat shock proteins Hsp70 and Hsp90 are required for RCNMV RNA replication and that they interact with p27, a virus-encoded component of the 480-kDa replicase complex, on the endoplasmic reticulum membrane. Using a cell-free viral translation/replication system in combination with specific inhibitors of Hsp70 and Hsp90, we found that inhibition of p27-Hsp70 interaction inhibits the formation of the 480-kDa complex but instead induces the accumulation of large complexes that are nonfunctional in viral RNA synthesis. In contrast, inhibition of p27-Hsp90 interaction did not induce such large complexes but rendered p27 incapable of binding to a specific viral RNA element, which is a critical step for the assembly of the 480-kDa replicase complex and viral RNA replication. Together, our results suggest that Hsp70 and Hsp90 regulate different steps in the assembly of the RCNMV replicase complex.

Template recognition mechanisms by replicase proteins differ between bipartite positive-strand genomic RNAs of a plant virus.

Recognition of RNA templates by viral replicase proteins is one of the key steps in the replication process of all RNA viruses. However, the mechanisms underlying this phenomenon, including primary RNA elements that are recognized by the viral replicase proteins, are not well understood. Here, we used aptamer pulldown assays with membrane fractionation and protein-RNA coimmunoprecipitation in a cell-free viral translation/replication system to investigate how viral replicase proteins recognize the bipartite genomic RNAs of the Red clover necrotic mosaic virus (RCNMV). RCNMV replicase proteins bound specifically to a Y-shaped RNA element (YRE) located in the 3' untranslated region (UTR) of RNA2, which also interacted with the 480-kDa replicase complexes that contain viral and host proteins. The replicase-YRE interaction recruited RNA2 to the membrane fraction. Conversely, RNA1 fragments failed to interact with the replicase proteins supplied in trans. The results of protein-RNA coimmunoprecipitation assays suggest that RNA1 interacts with the replicase proteins coupled with their translation. Thus, the initial template recognition mechanisms employed by the replicase differ between RCNMV bipartite genomic RNAs and RNA elements are primary determinants of the differential replication mechanism.

A versatile Tn7 transposon-based bioluminescence tagging tool for quantitative and spatial detection of bacteria in plants.

Investigation of plant-bacteria interactions requires quantification of in planta bacterial titers by means of cumbersome and time-consuming colony-counting assays. Here, we devised a broadly applicable tool for bioluminescence-based quantitative and spatial detection of bacteria in plants. We developed vectors that enable Tn7 transposon-mediated integration of the luxCDABE luciferase operon into a specific genomic location found ubiquitously across bacterial phyla. These vectors allowed for the generation of bioluminescent transformants of various plant pathogenic bacteria from the genera Pseudomonas, Rhizobium (Agrobacterium), and Ralstonia. Direct luminescence measurements of plant tissues inoculated with bioluminescent Pseudomonas syringae pv. tomato DC3000 (Pto-lux) reported bacterial titers as accurately as conventional colony-counting assays in Arabidopsis thaliana, Solanum lycopersicum, Nicotiana benthamiana, and Marchantia polymorpha. We further showed the usefulness of our vectors in converting previously generated Pto derivatives to isogenic bioluminescent strains. Importantly, quantitative bioluminescence assays using these Pto-lux strains accurately reported the effects of plant immunity and bacterial effectors on bacterial growth, with a dynamic range of four orders of magnitude. Moreover, macroscopic bioluminescence imaging illuminated the spatial patterns of Pto-lux growth in/on inoculated plant tissues. In conclusion, our vectors offer untapped opportunities to develop bioluminescence-based assays for a variety of plant-bacteria interactions.

Chitin-induced systemic disease resistance in rice requires both OsCERK1 and OsCEBiP and is mediated via perturbation of cell-wall biogenesis in leaves.

Chitin is a well-known elicitor of disease resistance and its recognition by plants is crucial to perceive fungal infections. Chitin can induce both a local immune response and a systemic disease resistance when provided as a supplement in soils. Unlike local immune responses, it is poorly explored how chitin-induced systemic disease resistance is developed. In this study, we report the systemic induction of disease resistance against the fungal pathogen Bipolaris oryzae by chitin supplementation of soils in rice. The transcriptome analysis uncovered genes related to cell-wall biogenesis, cytokinin signaling, regulation of phosphorylation, and defence priming in the development of chitin-induced systemic response. Alterations of cell-wall composition were observed in leaves of rice plants grown in chitin-supplemented soils, and the disease resistance against B. oryzae was increased in rice leaves treated with a cellulose biosynthesis inhibitor. The disruption of genes for lysin motif (LysM)-containing chitin receptors, OsCERK1 (Chitin elicitor receptor kinase 1) and OsCEBiP (Chitin elicitor-binding protein), compromised chitin-induced systemic disease resistance against B. oryzae and differential expression of chitin-induced genes found in wild-type rice plants. These findings suggest that chitin-induced systemic disease resistance in rice is caused by a perturbation of cell-wall biogenesis in leaves through long-distance signalling after local recognition of chitins by OsCERK1 and OsCEBiP.

Identification and characterization of the 480-kilodalton template-specific RNA-dependent RNA polymerase complex of red clover necrotic mosaic virus.

Replication of positive-strand RNA viruses occurs through the assembly of membrane-associated viral RNA replication complexes that include viral replicase proteins, viral RNA templates, and host proteins. Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA plant virus with a genome consisting of RNA1 and RNA2. The two proteins encoded by RNA1, a 27-kDa protein (p27) and an 88-kDa protein containing an RNA-dependent RNA polymerase (RdRP) motif (p88), are essential for RCNMV RNA replication. To analyze RCNMV RNA replication complexes, we used blue-native polyacrylamide gel electrophoresis (BN/PAGE), which enabled us to analyze detergent-solubilized large membrane protein complexes. p27 and p88 formed a complex of 480 kDa in RCNMV-infected plants. As a result of sucrose gradient sedimentation, the 480-kDa complex cofractionated with both endogenous template-bound and exogenous template-dependent RdRP activities. The amount of the 480-kDa complex corresponded to the activity of exogenous template-dependent RdRP, which produced RNA fragments by specifically recognizing the 3'-terminal core promoter sequences of RCNMV RNAs, but did not correspond to the activity of endogenous template-bound RdRP, which produced genome-sized RNAs without the addition of RNA templates. These results suggest that the 480-kDa complex contributes to template-dependent RdRP activities. We subjected those RdRP complexes to affinity purification and analyzed their components using two-dimensional BN/sodium dodecyl sulfate-PAGE (BN/SDS-PAGE) and mass spectrometry. The 480-kDa complex contained p27, p88, and possible host proteins, and the original affinity-purified RdRP preparation contained HSP70, HSP90, and several ribosomal proteins that were not detected in the 480-kDa complex. A model for the formation of RCNMV RNA replication complexes is proposed.