Sequence and Gene Expression Analysis of Recently Identified NLP from Plasmopara viticola

Abstract

Grapevine downy mildew, evoked by the obligate biotrophic oomycete Plasmopara viticola, is one of the most challenging diseases in viticulture. P. viticola establishes an infection by circumvention of plant immunity, which is achieved by the secretion of effector molecules. One family of potential effectors are the necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP). NLP are most abundant in plant pathogenic microorganisms and exist in cytotoxic and non-cyctotoxic forms. Cytotoxic NLP often act as virulence factors and are synthesized in necrotrophic or hemibiotrophic pathogens during the transition from biotrophic to necrotrophic growth. In addition to these cytotoxic NLP, many non-cytotoxic NLP have been identified; their function in biotrophic pathogens is still unknown. In 2020, eight different NLP coding genes were identified in P. viticola and named PvNLP1 to PvNLP8 (Plasmopara viticola NLP 1–8). In the present study, PvNLP4 to PvNLP8 were characterized by using qPCR analysis and transient expression in the model plant Nicotiana benthamiana. Gene expression analysis showed high PvNLP expression during the early stages of infection. Necrosis-inducing activity of PvNLP was not observed in the nonhost N. benthamiana.

A Comprehensive Assessment of the Secretome Responsible for Host Adaptation of the Legume Root Pathogen Aphanomyces euteiches

“…We confirmed the presence of GH62 (α-l-arabinofuranosidase), not reported in other oomycetes except in S. parasitica [41,76]. In addition to the large proportion of 'ricin' and 'necrosis-inducing' domains that are known to trigger cell necrosis [77][78][79], the core secretome contains adhe-sive and cysteine-rich proteins involved in oomycetes' pathogenicity, such as elicitin and CBM1 [30,80,81], or sterol binding CAP proteins that have recently been reported to be virulence factors in animal and plant pathogenic fungi [82].…”

“…known to trigger cell necrosis [77][78][79], the core secretome contains adhesive and cysteinerich proteins involved in oomycetes' pathogenicity, such as elicitin and CBM1 [30,80,81], or sterol binding CAP proteins that have recently been reported to be virulence factors in animal and plant pathogenic fungi [82]. In the 'non-core' secretome, the large majority of the secreted proteins displayed a predicted function that was already detected in the 'core' secretome.…”

Plasmopara viticola the Causal Agent of Downy Mildew of Grapevine: From Its Taxonomy to Disease Management

“…However, even while the RXLR motif was reported as responsible for effectors entering the host cell, its biological role remained controversial (Ellis and Dodds, 2011 ; Wawra et al, 2013 , 2017 ). Enzyme inhibitors, cysteine-rich proteins and Nep1-like proteins are located in a plant apoplast and interact with surface receptors and extracellular host targets (Kamoun, 2006 ; Askani et al, 2021 ). Another group of cytoplasmic effectors include crinkling and necrosis-inducing families (CRN) that are general for many oomycete members, in a contrast to RXLR that are specific to Phytophthora and species of downy mildews (Anderson et al, 2015 ; Lan et al, 2019 ).…”

Advances in understanding grapevine downy mildew: From pathogen infection to disease management

“…It was assumed that obligately biotrophic pathogens generally contained the noncytotoxic NLPs, as the biotrophs rely on living plant tissues for their growth and reproduction (Schumacher et al., 2020 ; Seidl & Van den Ackerveken, 2019 ). However, functional analyses of this kind of protein from obligate biotrophs were only performed on a couple of NLPs in P. viticola (Askani et al., 2021 ; Schumacher et al., 2020 ; Xiang et al., 2022 ) and H. arabidopsidis (Cabral et al., 2012 ). For example, a few NLP genes have been identified in P. viticola and most were highly expressed during the early stages of infection, suggesting that these genes may play major roles during pathogen penetration or initial colonization inside host tissues (Askani et al., 2021 ; Schumacher et al., 2020 ; Xiang et al., 2022 ).…”

“…However, functional analyses of this kind of protein from obligate biotrophs were only performed on a couple of NLPs in P. viticola (Askani et al., 2021 ; Schumacher et al., 2020 ; Xiang et al., 2022 ) and H. arabidopsidis (Cabral et al., 2012 ). For example, a few NLP genes have been identified in P. viticola and most were highly expressed during the early stages of infection, suggesting that these genes may play major roles during pathogen penetration or initial colonization inside host tissues (Askani et al., 2021 ; Schumacher et al., 2020 ; Xiang et al., 2022 ). However, whether PvNLP genes contribute to virulence for P. viticola is still unknown.…”