Nontoxic Nep1-Like Proteins of the Downy Mildew Pathogen Hyaloperonospora arabidopsidis: Repression of Necrosis-Inducing Activity by a Surface-Exposed Region

Abstract

The genome of the downy mildew pathogen Hyaloperonospora arabidopsidis encodes necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP). Although NLP are widely distributed in eukaryotic and prokaryotic plant pathogens, it was surprising to find these proteins in the obligate biotrophic oomycete H. arabidopsidis. Therefore, we analyzed the H. arabidopsidis NLP (HaNLP) family and identified 12 HaNLP genes and 15 pseudogenes. Most of the 27 genes form an H. arabidopsidis-specific cluster when compared with other oomycete NLP genes, suggesting this class of effectors has recently expanded in H. arabidopsidis. HaNLP transcripts were mainly detected during early infection stages. Agrobacterium tumefaciens-mediated transient expression and infiltration of recombinant NLP into tobacco and Arabidopsis leaves revealed that all HaNLP tested are noncytotoxic proteins. Even HaNLP3, which is most similar to necrosis-inducing NLP proteins of other oomycetes and which contains all amino acids that are critical for necrosis-inducing activity, did not induce necrosis. Chimeras constructed between HaNLP3 and the necrosis-inducing PsojNIP protein demonstrated that most of the HaNLP3 protein is functionally equivalent to PsojNIP, except for an exposed domain that prevents necrosis induction. The early expression and species-specific expansion of the HaNLP genes is suggestive of an alternative function of noncytolytic NLP proteins during biotrophic infection of plants.

Nep1-like proteins from three kingdoms of life act as a microbe-associated molecular pattern in Arabidopsis

“…H. arabidopsidis, the downy mildew pathogen of Arabidopsis, has an expanded family of 10 different NLP genes that encode noncytotoxic secreted proteins (26). To determine whether the HaNLPs would facilitate the infection process and enhance disease susceptibility of Arabidopsis, transgenic HaNLP overexpression lines were created.…”

“…As the observed activation of plant immunity in 35S:HaNLP-expressing plants could be the result of continuous overexpression, we created an estradiol-inducible line (containing an XVE:HaNLP3 construct). HaNLP3 was chosen for this as we studied this protein in more detail previously (26). A transgenic line was selected that showed no detectable HaNLP3 expression in untreated plants and a strong induction upon treatment with estradiol.…”

“…The discovery that noncytotoxic NLPs activate immunity in Arabidopsis was made while searching for a virulence function of these proteins in the downy mildew H. arabidopsidis (26). When transgenically expressed in Arabidopsis, 7 of the 10 HaNLPs induced severe growth reduction that resembled that of documented Arabidopsis autoimmune mutants, e.g., cpr1 and cpr5 (34,35), suggesting that the secreted proteins activate plant immunity.…”

“…Several plant-infecting oomycetes have large expansions of NLPs in their genomes (25)(26)(27), suggesting that these proteins play an important role in the pathogen's lifestyle. A clear virulence function was observed for NLP Pcc of the rot bacterium Pectobacterium carotovorum (27).…”

“…Five other NLP genes in this fungus encode noncytotoxic proteins (29), a phenomenon that is also observed in oomycetes. When tested by transient expression in tobacco, necrosis was only induced by 1 out of 3 tested NLPs of Phytophthora infestans (30), 8 out of 33 NLPs of Phytophthora sojae (31), whereas not a single 1 of 10 NLPs of Hyaloperonospora arabidopsidis tested caused necrosis (26). In contrast to cytotoxic NLPs that are mainly expressed during necrotrophic stages of infection, noncytotoxic NLPs appear to be expressed early during infection (26,30), suggesting they serve an, as-yet-unknown, function during penetration or initial colonization of the host.…”