Activation of innate immunity by membrane-localized receptors is conserved across eukaryotes. Plant genomes contain hundreds of such receptor-like genes and those encoding proteins with an ...extracellular leucine-rich repeat (LRR) domain represent the largest family. Here, we develop a high-throughput approach to study LRR receptor-like genes on a genome-wide scale. In total, 257 tobacco rattle virus-based constructs are generated to target 386 of the 403 identified LRR receptor-like genes in Nicotiana benthamiana for silencing. Using this toolkit, we identify the LRR receptor-like protein Response to XEG1 (RXEG1) that specifically recognizes the glycoside hydrolase 12 protein XEG1. RXEG1 associates with XEG1 via the LRR domain in the apoplast and forms a complex with the LRR receptor-like kinases BAK1 and SOBIR1 to transduce the XEG1-induced defense signal. Thus, this genome-wide silencing assay is demonstrated to be an efficient toolkit to pinpoint new immune receptors, which will contribute to developing durable disease resistance.
In plants, the apoplast is a critical battlefield for plant-microbe interactions. Plants secrete defense-related proteins into the apoplast to ward off the invasion of pathogens. How microbial ...pathogens overcome plant apoplastic immunity remains largely unknown. In this study, we reported that an atypical RxLR effector PsAvh181 secreted by Phytophthora sojae, inhibits the secretion of plant defense-related apoplastic proteins. PsAvh181 localizes to plant plasma membrane and essential for P. sojae infection. By co-immunoprecipitation assay followed by liquid chromatography-tandem mass spectrometry analyses, we identified the soybean GmSNAP-1 as a candidate host target of PsAvh181. GmSNAP-1 encodes a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein, which associates with GmNSF of the SNARE complex functioning in vesicle trafficking. PsAvh181 binds to GmSNAP-1 in vivo and in vitro. PsAvh181 interferes with the interaction between GmSNAP-1 and GmNSF, and blocks the secretion of apoplastic defense-related proteins, such as pathogenesis-related protein PR-1 and apoplastic proteases. Taken together, these data show that an atypical P. sojae RxLR effector suppresses host apoplastic immunity by manipulating the host SNARE complex to interfere with host vesicle trafficking pathway.
Extracellular vesicles (EVs) are important for cell-to-cell communication in animals. EVs also play important roles in plant-microbe interactions, but the underlying mechanisms remain elusive. Here, ...proteomic analyses of EVs from the soybean (Glycine max) root rot pathogen Phytophthora sojae identify the tetraspanin family proteins PsTET1 and PsTET3, which are recognized by Nicotiana benthamiana to trigger plant immune responses. Both proteins are required for the full virulence of P. sojae. The large extracellular loop (EC2) of PsTET3 is the key region recognized by N. benthamiana and soybean cells in a plant receptor-like kinase NbSERK3a/b dependent manner. TET proteins from oomycete and fungal plant pathogens are recognized by N. benthamiana thus inducing immune responses, whereas plant-derived TET proteins are not due to the sequence divergence of sixteen amino acids at the C-terminal of EC2. This feature allows plants to distinguish self and non-self EVs to trigger active defense responses against pathogenic eukaryotes.
Perception of pathogen-associated molecular patterns (PAMPs) often triggers various innate immune responses in plants. The transcriptional changes of defense-related genes are often used as a marker ...to assay PAMP-triggered plant immune response. Here we described a protocol to monitor the relative expression level of marker genes in
upon treatment with PAMPs. The procedure includes leaf treatment using PAMPs, total RNA isolation, cDNA synthesis, quantitative real-time PCR and data analysis. This protocol is applicable to monitor marker gene expression triggered by different PAMPs in
Currently, the research in memristor-based associative memory neural networks pays more attention to classical conditioning and lays less attention to operant conditioning. Moreover, a single ...reinforcing stimulus is applied in the most studies of operant conditioning. In this paper, a memristor-based duple-stimulus operant conditioning circuit is designed. The circuit utilizes duple stimuli which include two stimuli and realizes the effect of stimuli with two intensities on operant conditioning. In addition, the circuit is applied to a memristor-based neural network circuit of duple-reward and duple-punishment operant conditioning with time delay. The application circuit realizes duple-reward and duple-punishment operant conditioning with time delay on the basis of duple-stimulus operant conditioning circuit. Meanwhile, the factors that affect operant conditioning such as satiety and the immediacy of stimuli are implemented in the circuit. Different delays for duple-reward module and duple-punishment module are designed. The application circuit is closer to the practical application and is more consistent with biological characteristics. It provides more references for neural networks of operant conditioning with further development.
Diseases caused by Phytophthora pathogens devastate many crops worldwide. During infection, Phytophthora pathogens secrete effectors, which are central molecules for understanding the complex ...plant-Phytophthora interactions. In this study, we profiled the effector repertoire secreted by Phytophthora sojae into the soybean (Glycine max) apoplast during infection using liquid chromatography-mass spectrometry. A secreted aldose 1-epimerase (AEP1) was shown to induce cell death in Nicotiana benthamiana, as did the other two AEP1s from different Phytophthora species. AEP1 could also trigger immune responses in N. benthamiana, other Solanaceae plants, and Arabidopsis (Arabidopsis thaliana). A glucose dehydrogenase assay revealed AEP1 encodes an active AEP1. The enzyme activity of AEP1 is dispensable for AEP1-triggered cell death and immune responses, while AEP-triggered immune signaling in N. benthamiana requires the central immune regulator BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1. In addition, AEP1 acts as a virulence factor that mediates P. sojae extracellular sugar uptake by mutarotation of extracellular aldose from the α-anomer to the β-anomer. Taken together, these results revealed the function of a microbial apoplastic effector, highlighting the importance of extracellular sugar uptake for Phytophthora infection. To counteract, the key effector for sugar conversion can be recognized by the plant membrane receptor complex to activate plant immunity.
Summary
Leucine‐rich repeat receptor‐like kinases (LRR‐RLKs) comprise the largest class of membrane‐localized receptor‐like kinases in plants. Leucine‐rich repeat receptor‐like kinases are key immune ...sectors contributing to pattern‐triggered immunity (PTI), but whether LRR‐RLK mediates effector‐triggered immunity (ETI) in plants remains unclear.
In this study, we evaluated the function of LRR‐RLKs in regulating ETI by using a virus‐induced gene silencing (VIGS)‐based reverse genetic screening assay, and identified a LRR‐RLK named ETI‐dependent receptor‐like kinase 1 (EDK1) required for ETI triggered by the avirulence effector AVRblb2 secreted by Phytophthora infestans and its cognate receptor Rpi‐blb2. Silencing or knockout of EDK1 compromised immunity mediated by Rpi‐blb2 and the cell death triggered by recognition of AVRblb2.
NLR‐required for cell death 4 (NRC4), a signaling component acts downstream of Rpi‐blb2, was identified that interacts with EDK1 using the LC–MS analysis and the interaction was further evaluated by co‐immunoprecipitation. EDK1 promotes protein accumulation of NRC4 in a kinase‐dependent manner and positively regulates resistance to P. infestans in Nicotiana benthamiana.
Our study revealed that EDK1 positively regulates plant ETI through modulating accumulation of the NLR signaling component NRC4, representing a new regulatory role of the membrane‐localized LRR‐RLKs in plant immunity.
Elicitins are a large family of secreted proteins in Phytophthora. Clade 1 elicitins were identified decades ago as potent elicitors of immune responses in Nicotiana species, but the mechanisms ...underlying elicitin recognition are largely unknown. Here we identified an elicitin receptor in Nicotiana benthamiana that we named REL for Responsive to ELicitins. REL is a receptor-like protein (RLP) with an extracellular leucine-rich repeat (LRR) domain that mediates Phytophthora resistance by binding elicitins. Silencing or knocking out REL in N. benthamiana abolished elicitin-triggered cell death and immune responses. Domain deletion and site-directed mutagenesis revealed that the island domain (ID) located within the LRR domain of REL is crucial for elicitin recognition. In addition, sequence polymorphism in the ID underpins the genetic diversity of REL homologs in various Nicotiana species in elicitin recognition and binding. Remarkably, REL is phylogenetically distant from the elicitin response (ELR) protein, an LRR-RLP that was previously identified in the wild potato species Solanum microdontum and REL and ELR differ in the way they bind and recognize elicitins. Our findings provide insights into the molecular basis of plant innate immunity and highlight a convergent evolution of immune receptors towards perceiving the same elicitor.
Summary
Chitin is a structural and functional component of the fungal cell wall and also serves as a pathogen‐associated molecular pattern (PAMP) that triggers the innate immune responses of host ...plants. However, no or very little chitin is found in the fungus‐like oomycetes. In Phytophthora spp., the presence of chitin has not been demonstrated so far, although putative chitin synthase (CHS) genes, which encode the enzymes that synthesize chitin, are present in their genomes. Here, we revealed that chitin is present in the zoospores and released sporangia of Phytophthora, and this is most consistent with the transcriptional pattern of PcCHS in Phytophthora capsici and PsCHS1 in Phytophthora sojae. Disruption of the CHS genes indicated that PcCHS and PsCHS1, but not PsCHS2 (which exhibited very weak transcription), have similar functions involved in mycelial growth, sporangial production, zoospore release and the pathogenesis of P. capsici and P. sojae. We also suggest that chitin in the zoospores of P. capsici can act as a PAMP that is recognized by the chitin receptors AtLYK5 or AtCERK1 of Arabidopsis. These results provide new insights into the biological significance of chitin and CHSs in Phytophthora and help with the identification of potential targets for disease control.
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