The novel peptide NbPPI1 identified from Nicotiana benthamiana triggers immune responses and enhances resistance against Phytophthora pathogens.

In plants, recognition of small secreted peptides, such as damage/danger-associated molecular patterns (DAMPs), regulates diverse processes, including stress and immune responses. Here, we identified an SGPS (Ser-Gly-Pro-Ser) motif-containing peptide, Nicotiana tabacum NtPROPPI, and its two homologs in Nicotiana benthamiana, NbPROPPI1 and NbPROPPI2. Phytophthora parasitica infection and salicylic acid (SA) treatment induced NbPROPPI1/2 expression. Moreover, SignalP predicted that the 89-amino acid NtPROPPI includes a 24-amino acid N-terminal signal peptide and NbPROPPI1/2-GFP fusion proteins were mainly localized to the periplasm. Transient expression of NbPROPPI1/2 inhibited P. parasitica colonization, and NbPROPPI1/2 knockdown rendered plants more susceptible to P. parasitica. An eight-amino-acid segment in the NbPROPPI1 C-terminus was essential for its immune function and a synthetic 20-residue peptide, NbPPI1, derived from the C-terminus of NbPROPPI1 provoked significant immune responses in N. benthamiana. These responses led to enhanced accumulation of reactive oxygen species, activation of mitogen-activated protein kinases, and up-regulation of the defense genes Flg22-induced receptor-like kinase (FRK) and WRKY DNA-binding protein 33 (WRKY33). The NbPPI1-induced defense responses require Brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1). These results suggest that NbPPI1 functions as a DAMP in N. benthamiana; this novel DAMP provides a potentially useful target for improving plant resistance to Pytophthora pathogens.

Cytidine-to-Uridine RNA Editing Factor NbMORF8 Negatively Regulates Plant Immunity to Phytophthora Pathogens.

Mitochondria and chloroplasts play key roles in plant-pathogen interactions. Cytidine-to-uridine (C-to-U) RNA editing is a critical posttranscriptional modification in mitochondria and chloroplasts that is specific to flowering plants. Multiple organellar RNA-editing factors (MORFs) form a protein family that participates in C-to-U RNA editing, but little is known regarding their immune functions. Here, we report the identification of NbMORF8, a negative regulator of plant immunity to Phytophthora pathogens. Using virus-induced gene silencing and transient expression in Nicotiana benthamiana, we show that NbMORF8 functions through the regulation of reactive oxygen species production, salicylic acid signaling, and accumulation of multiple Arg-X-Leu-Arg effectors of Phytophthora pathogens. NbMORF8 is localized to mitochondria and chloroplasts, and its immune function requires mitochondrial targeting. The conserved MORF box domain is not required for its immune function. Furthermore, we show that the preferentially mitochondrion-localized NbMORF proteins negatively regulate plant resistance against Phytophthora, whereas the preferentially chloroplast-localized ones are positive immune regulators. Our study reveals that the C-to-U RNA-editing factor NbMORF8 negatively regulates plant immunity to the oomycete pathogen Phytophthora and that mitochondrion- and chloroplast-localized NbMORF family members exert opposing effects on immune regulation.

The Arabidopsis thaliana gene AtERF019 negatively regulates plant resistance to Phytophthora parasitica by suppressing PAMP-triggered immunity.

Phytophthora species are destructive plant pathogens that cause significant crop losses worldwide. To understand plant susceptibility to oomycete pathogens and to explore novel disease resistance strategies, we employed the Arabidopsis thaliana-Phytophthora parasitica model pathosystem and screened for A. thaliana T-DNA insertion mutant lines resistant to P. parasitica. This led to the identification of the resistant mutant 267-31, which carries two T-DNA insertion sites in the promoter region of the ethylene-responsive factor 19 gene (ERF019). Quantitative reverse transcription PCR (RT-qPCR) assays showed that the expression of ERF019 was induced during P. parasitica infection in the wild type, which was suppressed in the 267-31 mutant. Additional erf019 mutants were generated using CRISPR/Cas9 technology and were confirmed to have increased resistance to P. parasitica. In contrast, ERF019 overexpression lines were more susceptible. Transient overexpression assays in Nicotiana benthamiana showed that the nuclear localization of ERF019 is crucial for its susceptible function. RT-qPCR analyses showed that the expression of marker genes for multiple defence pathways was significantly up-regulated in the mutant compared with the wild type during infection. Flg22-induced hydrogen peroxide accumulation and reactive oxygen species burst were impaired in ERF019 overexpression lines, and flg22-induced MAPK activation was enhanced in erf019 mutants. Moreover, transient overexpression of ERF019 strongly suppressed INF-triggered cell death in N. benthamiana. These results reveal the importance of ERF019 in mediating plant susceptibility to P. parasitica through suppression of pathogen-associated molecular pattern-triggered immunity.

A Phytophthora capsici RXLR Effector Targets and Inhibits a Plant PPIase to Suppress Endoplasmic Reticulum-Mediated Immunity.

Phytophthora pathogens secrete a large arsenal of effectors that manipulate host processes to create an environment conducive to pathogen colonization. However, the underlying mechanisms by which Phytophthora effectors manipulate host plant cells still remain largely unclear. In this study, we report that PcAvr3a12, a Phytophthora capsici RXLR effector and a member of the Avr3a effector family, suppresses plant immunity by targeting and inhibiting host plant peptidyl-prolyl cis-trans isomerase (PPIase). Overexpression of PcAvr3a12 in Arabidopsis thaliana enhanced plant susceptibility to P. capsici. FKBP15-2, an endoplasmic reticulum (ER)-localized protein, was identified as a host target of PcAvr3a12 during early P. capsici infection. Analyses of A. thaliana T-DNA insertion mutant (fkbp15-2), RNAi, and overexpression lines consistently showed that FKBP15-2 positively regulates plant immunity in response to Phytophthora infection. FKBP15-2 possesses PPIase activity essential for its contribution to immunity but is directly suppressed by PcAvr3a12. Interestingly, we found that FKBP15-2 is involved in ER stress sensing and is required for ER stress-mediated plant immunity. Taken together, these results suggest that P. capsici deploys an RXLR effector, PcAvr3a12, to facilitate infection by targeting and suppressing a novel ER-localized PPIase, FKBP15-2, which is required for ER stress-mediated plant immunity.

Phenotypic and genetic characterization of resistance in Arabidopsis thaliana to the oomycete pathogen Phytophthora parasitica.

The interaction between Arabidopsis thaliana and the oomycete pathogen Phytophthora parasitica emerges as a model for exploring the molecular basis and evolution of recognition and host defense. Phenotypic variation and genetic analysis is essential to dissect the underlying mechanisms in plant-oomycete interaction. In this study, the reaction phenotypes of 28 A. thaliana accessions to P. parasitica strain Pp016 were examined using detached leaf infection assay. The results showed the presence of four distinct groups based on host response and disease development. Of all the accessions examined, Zurich (Zu-1) is highly resistant to P. parasitica. Microscopic characterization showed that rapid and severe hypersensitive response at the primary infection epidermal cells is associated with disease resistance. Furthermore, Zu-1 is resistant to a set of 20 diverse P. parasitica strains, which were collected from different host plants and exhibited differential specificities on a set of tobacco cultivars. However, Zu-1 is susceptible to P. parasitica when the root is inoculated, suggesting differential expression of associated resistance genes in the root and foliar tissues. Genetic analysis by crossing Zu-1 and the susceptible accession Landsberg (Ler) showed that the resistance in Zu-1 to P. parasitica is semi-dominant, as shown by infection assays of F1 progenies, and is likely conferred by a single locus, defined as RPPA1 (Zu-1) (for Resistance to P. parasitica 1), as shown by analysis of F2 segregating populations. By employing specific-locus amplified fragment sequencing (SLAF-seq) strategy to identify molecular markers potentially linked to the locus, the strongest associated region was determined to be located between 7.1 and 11.2 Mb in chromosome IV. The future cloning of RPPA1 (Zu-1) locus will facilitate improved understanding of plant broad-spectrum disease resistance to oomycete pathogens.