A Small Cysteine-Rich Phytotoxic Protein of Phytophthora capsici Functions as Both Plant Defense Elicitor and Virulence Factor.

Small cysteine-rich (SCR) proteins, including fungal avirulence proteins, play important roles in pathogen-plant interactions. SCR protein-encoding genes have been discovered in the genomes of Phytophthora pathogens but their functions during pathogenesis remain obscure. Here, we report the characterization of one Phytophthora capsici SCR protein (namely, SCR82) with similarity to Phytophthora cactorum phytotoxic protein PcF. The scr82 gene has 10 allelic sequences in the P. capsici population. Homologs of SCR82 were not identified in fungi or other organisms but in Phytophthora relative species. Initially, scr82 was weakly expressed during the mycelium, sporangium, and zoospore stages but quickly upregulated when the infection initiated. Both ectopic expression of SCR82 and recombinant yeast-expressed protein (rSCR82) caused cell death on tomato leaves. Upon treatment, rSCR82 induced plant defense responses, including the induction of defense gene expression, reactive oxygen species burst, and callose deposition. Knockout of scr82 in P. capsici by CRISPR/Cas9 severely impaired its virulence on host plants and significantly reduced its resistance against oxidative stress. Inversely, its overexpression increased the pathogen's virulence and tolerance to oxidative stress. Our results collectively demonstrate that SCR82 functions as both an important virulence factor and plant defense elicitor, which is conserved across Phytophthora spp.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The RXLR Effector PcAvh1 Is Required for Full Virulence of Phytophthora capsici.

Plant pathogens employ diverse secreted effector proteins to manipulate host physiology and defense in order to foster diseases. The destructive Phytophthora pathogens encode hundreds of cytoplasmic effectors, which are believed to function inside the plant cells. Many of these cytoplasmic effectors contain the conserved N-terminal RXLR motif. Understanding the virulence function of RXLR effectors will provide important knowledge of Phytophthora pathogenesis. Here, we report the characterization of RXLR effector PcAvh1 from the broad-host range pathogen Phytophthora capsici. Only expressed during infection, PcAvh1 is quickly induced at the early infection stages. CRISPR/Cas9-knockout of PcAvh1 in P. capsici severely impairs virulence while overexpression enhances disease development in Nicotiana benthamiana and bell pepper, demonstrating that PcAvh1 is an essential virulence factor. Ectopic expression of PcAvh1 induces cell death in N. benthamiana, tomato, and bell pepper. Using yeast two-hybrid screening, we found that PcAvh1 interacts with the scaffolding subunit of the protein phosphatase 2A (PP2Aa) in plant cells. Virus-induced gene silencing of PP2Aa in N. benthamiana attenuates resistance to P. capsici and results in dwarfism, suggesting that PP2Aa regulates plant immunity and growth. Collectively, these results suggest that PcAvh1 contributes to P. capsici infection, probably through its interaction with host PP2Aa.

Identification and functional analysis of the NLP-encoding genes from the phytopathogenic oomycete Phytophthora capsici.

Phytophthora capsici is a hemibiotrophic, phytopathogenic oomycete that infects a wide range of crops, resulting in significant economic losses worldwide. By means of a diverse arsenal of secreted effector proteins, hemibiotrophic pathogens may manipulate plant cell death to establish a successful infection and colonization. In this study, we described the analysis of the gene family encoding necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) in P. capsici, and identified 39 real NLP genes and 26 NLP pseudogenes. Out of the 65 predicted NLP genes, 48 occur in groups with two or more genes, whereas the remainder appears to be singletons distributed randomly among the genome. Phylogenetic analysis of the 39 real NLPs delineated three groups. Key residues/motif important for the effector activities are degenerated in most NLPs, including the nlp24 peptide consisting of the conserved region I (11-aa immunogenic part) and conserved region II (the heptapeptide GHRHDWE motif) that is important for phytotoxic activity. Transcriptional profiling of eight selected NLP genes indicated that they were differentially expressed during the developmental and plant infection phases of P. capsici. Functional analysis of ten cloned NLPs demonstrated that Pc11951, Pc107869, Pc109174 and Pc118548 were capable of inducing cell death in the Solanaceae, including Nicotiana benthamiana and hot pepper. This study provides an overview of the P. capsici NLP gene family, laying a foundation for further elucidating the pathogenicity mechanism of this devastating pathogen.

Transcription profiling and identification of infection-related genes in Phytophthora cactorum.

Phytophthora cactorum, an oomycete pathogen, infects more than 200 plant species within several plant families. To gain insight into the repertoire of the infection-related genes of P. cactorum, Illumina RNA-Seq was used to perform a global transcriptome analysis of three life cycle stages of the pathogen, mycelia (MY), zoospores (ZO) and germinating cysts with germ tubes (GC). From over 9.8 million Illumina reads for each library, 18,402, 18,569 and 19,443 distinct genes were identified for MY, ZO and GC libraries, respectively. Furthermore, the transcriptome difference among MY, ZO and GC stages was investigated. Gene ontology (GO) and KEGG pathway enrichment analyses revealed diverse biological functions and processes. Comparative analysis identified a large number of genes that are associated with specific stages and pathogenicity, including 166 effector genes. Of them, most of RXLR and NLP genes showed induction while the majority of CRN genes were down-regulated in GC, the important pre-infection stage, compared to either MY or ZO. And 14 genes encoding small cysteine-rich (SCR) secretory proteins showed differential expression during the developmental stages and in planta. Ectopic expression in the Solanaceae indicated that SCR113 and one elicitin PcINF1 can trigger cell death on Nicotiana benthamiana, tobacco (N. tabacum) and tomato (Solanum lycopersicum) leaves. Neither conserved domain nor homologues of SCR113 in other organisms can be identified. Collectively, our study provides a comprehensive examination of gene expression across three P. cactorum developmental stages and describes pathogenicity-related genes, all of which will help elucidate the pathogenicity mechanism of this destructive pathogen.