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Phytophthora sojae, an oomycete pathogen, produces a large number of effector proteins that enter into host cells. The Crinklers (Crinkling and Necrosis, CRN) are cytoplasmic effectors that are conserved in oomycete pathogens and their encoding genes are highly expressed at the infective stages in P. sojae. However, their roles in pathogenesis are largely unknown. Here, we functionally characterized an effector PsCRN70 by transiently and stably overexpressing it in Nicotiana benthamiana. We demonstrated that PsCRN70 was localized to the plant cell nucleus and suppressed cell death elicited by all the tested cell death-inducing proteins, including BAX, PsAvh241, PsCRN63, PsojNIP and R3a/Avr3a. Overexpression of the PsCRN70 gene in N. benthamiana enhanced susceptibility to P. parasitica. The H2O2 accumulation in the PsCRN70-transgenic plants was reduced compared to the GFP-lines. The transcriptional levels of the defense-associated genes, including PR1b, PR2b, ERF1 and LOX, were also down-regulated in the PsCRN70-transgenic lines. Our results suggest that PsCRN70 may function as a universal suppressor of the cell death induced by many elicitors, the host H2O2 accumulation and the expression of defense-associated genes, and therefore promotes pathogen infection. [ABSTRACT FROM AUTHOR]

Phytophthora and other oomycetes secrete a large number of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling and necrosis inducing proteins (CRN), or Crinkler. Here, we first investigated the evolutionary patterns and mechanisms of CRN effectors in Phytophthora sojae and compared them to two other Phytophthora species. The genes encoding CRN effectors could be divided into 45 orthologous gene groups (OGG), and most OGGs unequally distributed in the three species, in which each underwent large number of gene gains or losses, indicating that the CRN genes expanded after species evolution in Phytophthora and evolved through pathoadaptation. The 134 expanded genes in P. sojae encoded family proteins including 82 functional genes and expressed at higher levels while the other 68 genes encoding orphan proteins were less expressed and contained 50 pseudogenes. Furthermore, we demonstrated that most expanded genes underwent gene duplication or/and fragment recombination. Three different mechanisms that drove gene duplication or recombination were identified. Finally, the expanded CRN effectors exhibited varying pathogenic functions, including induction of programmed cell death (PCD) and suppression of PCD through PAMP-triggered immunity or/and effector-triggered immunity. Overall, these results suggest that gene duplication and fragment recombination may be two mechanisms that drive the expansion and neofunctionalization of the CRN family in P. sojae, which aids in understanding the roles of CRN effectors within each oomycete pathogen. [ABSTRACT FROM AUTHOR]

Identification of pathogen-inducible promoters largely lags behind cloning of the genes for disease resistance. Here, we cloned the soybean GmaPPO12 gene and found that it was rapidly and strongly induced by Phytophthora sojae infection. Computational analysis revealed that its promoter contained many known cis-elements, including several defense related transcriptional factor-binding boxes. We showed that the promoter could mediate induction of GUS expression upon infection in both transient expression assays in Nicotiana benthamiana and stable transgenic soybean hairy roots. Importantly, we demonstrated that pathogen-induced expression of the GmaPPO12 promoter was higher than that of the soybean GmaPR1a promoter. A progressive 5’ and 3’ deletion analysis revealed two fragments that were essential for promoter activity. Thus, the cloned promoter could be used in transgenic plants to enhance resistance to phytophthora pathogens, and the identified fragment could serve as a candidate to produce synthetic pathogen-induced promoters. [ABSTRACT FROM AUTHOR]

Phytophthora sojae, an oomycete pathogen, produces a large number of effector proteins that enter into host cells. The Crinklers (Crinkling and Necrosis, CRN) are cytoplasmic effectors that are conserved in oomycete pathogens and their encoding genes are highly expressed at the infective stages in P. sojae. However, their roles in pathogenesis are largely unknown. Here, we functionally characterized an effector PsCRN70 by transiently and stably overexpressing it in Nicotiana benthamiana. We demonstrated that PsCRN70 was localized to the plant cell nucleus and suppressed cell death elicited by all the tested cell death-inducing proteins, including BAX, PsAvh241, PsCRN63, PsojNIP and R3a/Avr3a. Overexpression of the PsCRN70 gene in N. benthamiana enhanced susceptibility to P. parasitica. The H2O2 accumulation in the PsCRN70-transgenic plants was reduced compared to the GFP-lines. The transcriptional levels of the defense-associated genes, including PR1b, PR2b, ERF1 and LOX, were also down-regulated in the PsCRN70-transgenic lines. Our results suggest that PsCRN70 may function as a universal suppressor of the cell death induced by many elicitors, the host H2O2 accumulation and the expression of defense-associated genes, and therefore promotes pathogen infection. [ABSTRACT FROM AUTHOR]

Phytophthora and other oomycetes secrete a large number of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling and necrosis inducing proteins (CRN), or Crinkler. Here, we first investigated the evolutionary patterns and mechanisms of CRN effectors in Phytophthora sojae and compared them to two other Phytophthora species. The genes encoding CRN effectors could be divided into 45 orthologous gene groups (OGG), and most OGGs unequally distributed in the three species, in which each underwent large number of gene gains or losses, indicating that the CRN genes expanded after species evolution in Phytophthora and evolved through pathoadaptation. The 134 expanded genes in P. sojae encoded family proteins including 82 functional genes and expressed at higher levels while the other 68 genes encoding orphan proteins were less expressed and contained 50 pseudogenes. Furthermore, we demonstrated that most expanded genes underwent gene duplication or/and fragment recombination. Three different mechanisms that drove gene duplication or recombination were identified. Finally, the expanded CRN effectors exhibited varying pathogenic functions, including induction of programmed cell death (PCD) and suppression of PCD through PAMP-triggered immunity or/and effector-triggered immunity. Overall, these results suggest that gene duplication and fragment recombination may be two mechanisms that drive the expansion and neofunctionalization of the CRN family in P. sojae, which aids in understanding the roles of CRN effectors within each oomycete pathogen. [ABSTRACT FROM AUTHOR]