Abstract
Phytophthora sojae encodes hundreds of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling- and necrosis-inducing proteins (CRN) or Crinkler. Their functions and mechanisms in pathogenesis are mostly unknown. Here, we identify a group of five P. sojae-specific CRN-like genes with high levels of sequence similarity, of which three are putative pseudogenes. Functional analysis shows that the two functional genes encode proteins with predicted nuclear localization signals that induce contrasting responses when expressed in Nicotiana benthamiana and soybean (Glycine max). PsCRN63 induces cell death, while PsCRN115 suppresses cell death elicited by the P. sojae necrosis-inducing protein (PsojNIP) or PsCRN63. Expression of CRN fragments with deleted signal peptides and FLAK motifs demonstrates that the carboxyl-terminal portions of PsCRN63 or PsCRN115 are sufficient for their activities. However, the predicted nuclear localization signal is required for PsCRN63 to induce cell death but not for PsCRN115 to suppress cell death. Furthermore, silencing of the PsCRN63 and PsCRN115 genes in P. sojae stable transformants leads to a reduction of virulence on soybean. Intriguingly, the silenced transformants lose the ability to suppress host cell death and callose deposition on inoculated plants. These results suggest a role for CRN effectors in the suppression of host defense responses.
Highlights
Phytophthora sojae encodes hundreds of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling- and necrosis-inducing proteins (CRN) or Crinkler
We selected one group of genes for functional analysis for three reasons: first, the predicted gene models encode proteins with a predicted nuclear localization signals (NLS) (Fig. 1; Cokol et al, 2000); second, the group shows high sequence similarity with P. infestans CRN2 (PiCRN2), which has the ability to elicit cell death in N. benthamiana and induce the expression of defense responses in tomato (Solanum lycopersicum; Torto et al, 2003); and third, selected genes within the group are the most highly expressed among all P. sojae RXLR and CRN effectors based upon transcriptional profiling of various life cycle and infection stages
We have examined two CRN effectors of P. sojae with high sequence similarity and high expression levels
Summary
Phytophthora sojae encodes hundreds of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling- and necrosis-inducing proteins (CRN) or Crinkler. Their functions and mechanisms in pathogenesis are mostly unknown. Many plant pathogens, including bacteria, fungi, oomycetes, and nematodes, secrete distinct proteins into different cellular compartments of their hosts to modulate host defense circuitry and benefit parasite colonization (Bhavsar et al, 2007; Hogenhout et al, 2009; Tyler, 2009). The common pattern, containing a signal peptide and a conserved RXLR-dEER motif, was shown to be responsible for delivering proteins into host plant cells colonized by P. infestans (Whisson et al, 2007) or P. sojae (Dou et al, 2008b) and is presumed to have the same function in most oomycetes. Once delivered inside host cells, RXLR effectors are involved in the suppression of host basal resistance (Sohn et al, 2007) and cell death (Bos et al, 2006; Dou et al, 2008a) when the cognate R genes are absent
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