Plant receptor-like protein activation by a microbial glycoside hydrolase.

Plants rely on cell-surface-localized pattern recognition receptors to detect pathogen- or host-derived danger signals and trigger an immune response1-6. Receptor-like proteins (RLPs) with a leucine-rich repeat (LRR) ectodomain constitute a subgroup of pattern recognition receptors and play a critical role in plant immunity1-3. Mechanisms underlying ligand recognition and activation of LRR-RLPs remain elusive. Here we report a crystal structure of the LRR-RLP RXEG1 from Nicotiana benthamiana that recognizes XEG1 xyloglucanase from the pathogen Phytophthora sojae. The structure reveals that specific XEG1 recognition is predominantly mediated by an amino-terminal and a carboxy-terminal loop-out region (RXEG1(ID)) of RXEG1. The two loops bind to the active-site groove of XEG1, inhibiting its enzymatic activity and suppressing Phytophthora infection of N. benthamiana. Binding of XEG1 promotes association of RXEG1(LRR) with the LRR-type co-receptor BAK1 through RXEG1(ID) and the last four conserved LRRs to trigger RXEG1-mediated immune responses. Comparison of the structures of apo-RXEG1(LRR), XEG1-RXEG1(LRR) and XEG1-BAK1-RXEG1(LRR) shows that binding of XEG1 induces conformational changes in the N-terminal region of RXEG1(ID) and enhances structural flexibility of the BAK1-associating regions of RXEG1(LRR). These changes allow fold switching of RXEG1(ID) for recruitment of BAK1(LRR). Our data reveal a conserved mechanism of ligand-induced heterodimerization of an LRR-RLP with BAK1 and suggest a dual function for the LRR-RLP in plant immunity.

AtRTP5 negatively regulates plant resistance to Phytophthora pathogens by modulating the biosynthesis of endogenous jasmonic acid and salicylic acid.

Plants have evolved powerful immune systems to recognize pathogens and avoid invasions, but the genetic basis of plant susceptibility is less well-studied, especially to oomycetes, which cause disastrous diseases in many ornamental plants and food crops. In this research, we identified a negative regulator of plant immunity to the oomycete Phytophthora parasitica, AtRTP5 (Arabidopsis thaliana Resistant to Phytophthora 5), which encodes a WD40 repeat domain-containing protein. The AtRTP5 protein, which was tagged with green fluorescent protein (GFP), is localized in the nucleus and plasma membrane. Both the A. thaliana T-DNA insertion rtp5 mutants and the Nicotiana benthamiana RTP5 (NbRTP5) silencing plants showed enhanced resistance to P. parasitica, while overexpression of AtRTP5 rendered plants more susceptible. The transcriptomic analysis showed that mutation of AtRTP5 suppressed the biosynthesis of endogenous jasmonic acid (JA) and JA-dependent responses. In contrast, salicylic acid (SA) biosynthesis and SA-dependent responses were activated in the T-DNA insertion mutant rtp5-3. These results show that AtRTP5 acts as a conserved negative regulator of plant immunity to Phytophthora pathogens by interfering with JA and SA signalling pathways.

Simultaneous transcriptome analysis of oil palm clones and Phytophthora palmivora reveals oil palm defense strategies.

Phytophthora palmivora is an oomycete that causes oil palm bud rot disease. To understand the molecular mechanisms of this disease, palm clones with contrasting responses (Ortet 34, resistant and Ortet 57, susceptible) were inoculated with P. palmivora, and RNAseq gene expression analysis was performed. The transcriptome was obtained by sequencing using Illumina HiSeq2500 technology during the asymptomatic phase (24, 72 and 120 hours postinfection, hpi). A simultaneous analysis of differentially expressed gene (DEG) profiles in palm and P. palmivora was carried out. Additionally, Gene Ontology (GO) and gene network analysis revealed differences in the transcriptional profile of the two ortets, where a high specificity of the pathogen to colonize the susceptible ortet was found. The transcriptional analysis provided an overview of the genes involved in the recognition and signaling of this pathosystem, where different transcription factors, phytohormones, proteins associated with cell wall hardening and nitrogen metabolism contribute to the resistance of oil palm to P. palmivora. This research provides a description of the molecular response of oil palm to P. palmivora, thus becoming an important source of molecular markers for the study of genotypes resistant to bud rot disease.

An Oomycete CRN Effector Reprograms Expression of Plant HSP Genes by Targeting their Promoters.

Oomycete pathogens produce a large number of CRN effectors to manipulate plant immune responses and promote infection. However, their functional mechanisms are largely unknown. Here, we identified a Phytophthora sojae CRN effector PsCRN108 which contains a putative DNA-binding helix-hairpin-helix (HhH) motif and acts in the plant cell nucleus. Silencing of the PsCRN108 gene reduced P. sojae virulence to soybean, while expression of the gene in Nicotiana benthamiana and Arabidopsis thaliana enhanced plant susceptibility to P. capsici. Moreover, PsCRN108 could inhibit expression of HSP genes in A. thaliana, N. benthamiana and soybean. Both the HhH motif and nuclear localization signal of this effector were required for its contribution to virulence and its suppression of HSP gene expression. Furthermore, we found that PsCRN108 targeted HSP promoters in an HSE- and HhH motif-dependent manner. PsCRN108 could inhibit the association of the HSE with the plant heat shock transcription factor AtHsfA1a, which initializes HSP gene expression in response to stress. Therefore, our data support a role for PsCRN108 as a nucleomodulin in down-regulating the expression of plant defense-related genes by directly targeting specific plant promoters.

The Activation of Phytophthora Effector Avr3b by Plant Cyclophilin is Required for the Nudix Hydrolase Activity of Avr3b.

Plant pathogens secrete an arsenal of effector proteins to impair host immunity. Some effectors possess enzymatic activities that can modify their host targets. Previously, we demonstrated that a Phytophthora sojae RXLR effector Avr3b acts as a Nudix hydrolase when expressed in planta; and this enzymatic activity is required for full virulence of P. sojae strain P6497 in soybean (Glycine max). Interestingly, recombinant Avr3b produced by E. coli does not have the hydrolase activity unless it was incubated with plant protein extracts. Here, we report the activation of Avr3b by a prolyl-peptidyl isomerase (PPIase), cyclophilin, in plant cells. Avr3b directly interacts with soybean cyclophilin GmCYP1, which activates the hydrolase activity of Avr3b in a PPIase activity-dependent manner. Avr3b contains a putative Glycine-Proline (GP) motif; which is known to confer cyclophilin-binding in other protein substrates. Substitution of the Proline (P132) in the putative GP motif impaired the interaction of Avr3b with GmCYP1; as a result, the mutant Avr3bP132A can no longer be activated by GmCYP1, and is also unable to promote Phytophthora infection. Avr3b elicits hypersensitive response (HR) in soybean cultivars producing the resistance protein Rps3b, but Avr3bP132A lost its ability to trigger HR. Furthermore, silencing of GmCYP1 rendered reduced cell death triggered by Avr3b, suggesting that GmCYP1-mediated Avr3b maturation is also required for Rps3b recognition. Finally, cyclophilins of Nicotiana benthamiana can also interact with Avr3b and activate its enzymatic activity. Overall, our results demonstrate that cyclophilin is a "helper" that activates the enzymatic activity of Avr3b after it is delivered into plant cells; as such, cyclophilin is required for the avirulence and virulence functions of Avr3b.

beta-aminobutyric acid primes an NADPH oxidase-dependent reactive oxygen species production during grapevine-triggered immunity.

The molecular mechanisms underlying the process of priming are poorly understood. In the present study, we investigated the early signaling events triggered by beta-aminobutyric acid (BABA), a well-known priming-mediated plant resistance inducer. Our results indicate that, in contrast to oligogalacturonides (OG), BABA does not elicit typical defense-related early signaling events nor defense-gene expression in grapevine. However, in OG-elicited cells pretreated with BABA, production of reactive oxygen species (ROS) and expression of the respiratory-burst oxidase homolog RbohD gene were primed. In response to the causal agent of downy mildew Plasmopara viticola, a stronger ROS production was specifically observed in BABA-treated leaves. This process was correlated with an increased resistance. The NADPH oxidase inhibitor diphenylene iodonium (DPI) abolished this primed ROS production and reduced the BABA-induced resistance (BABA-IR). These results suggest that priming of an NADPH oxidase-dependent ROS production contributes to BABA-IR in the Vitis-Plasmopara pathosystem.

Production of monoclonal antibodies against peripheral-vesicle proteins in zoospores of Phytophthora nicotianae.

A coimmunisation protocol using microsomal fractions from Phytophthora nicotianae cells has enhanced the production of monoclonal antibodies directed towards proteins produced during asexual sporulation. Over 40% of the antibodies targeted three categories of zoospore peripheral vesicles. Five antibodies label the contents of dorsal vesicles, with three of these reacting with two P. nicotianae polypeptides with a relative molecular mass of approximately 100 kDa. Two antibodies label the contents of large peripheral vesicles and react with two very high-molecular-weight polypeptides in extracts of P. nicotianae cells. These antibodies cross-react with the contents of large peripheral vesicles in P. cinnamomi zoospores. Ten antibodies label the contents of P. nicotianae zoospore ventral vesicles and react with a single polypeptide with a relative molecular mass of 230 kDa. A number of these antibodies against the contents of ventral vesicles in P. nicotianae zoospores cross-react with ventral-vesicle proteins in P. cinnamomi cells in immunofluorescence and immunoblot assays. The study illustrates the value of the coimmunisation protocol and has produced antibodies that could be instrumental in the cloning of genes encoding peripheral-vesicle proteins.

Altered lignin structure and resistance to pathogens in spi 2-expressing tobacco plants.

The physiological role of the Norway spruce [ Picea abies (L.) Karst.] spi 2 gene, encoding a defense-related cationic peroxidase was examined in transgenic tobacco (Nicotiana tabacum L.). Expression of spi 2, under control of the 35S promoter, in tobacco plants resulted in higher total peroxidase activities. The phenotype of the spi 2-transformed lines was normal. The spi 2-transformed lines displayed lignin levels similar to levels in the control line, but with some alteration in lignin histochemistry and structure. These changes were associated with reduced flexibility of the tobacco stems. The defense against pathogenic microorganisms was altered in the transgenic tobacco plants compared with control plants. High peroxidase activities increased the susceptibility to the pathogenic oomycete Phytophthora parasitica var. nicotianae, but increased the ability of the tobacco plants to suppress growth of the pathogenic bacterium Erwinia carotovora.