Transcriptional plasticity enables oomycetes to rapidly adapt to environmental challenges including emerging host resistance. For example, the soybean pathogen Phytophthora sojae can overcome resistance conferred by the host resistance gene Rps1b through natural silencing of its corresponding effector gene, Avr1b-1. With the Phytophthora CRISPR/Cas9 genome editing system, it is possible to generate site-specific knock-out (KO) and knock-in (KI) mutants and to investigate the biological functions of target genes. In this study, the Avr1b-1 gene was deleted from the P. sojae genome using a homology-directed recombination strategy that replaced Avr1b-1 with a gene encoding the fluorescent protein mCherry. As expected, all selected KO transformants gained virulence on Rps1b plants, while infection of plants lacking Rps1b was not compromised. When a sgRNA-resistant version of Avr1b-1 was reintroduced into the Avr1b-1 locus of an Avr1b KO transformant, KI transformants with a well-transcribed Avr1b-1 gene were unable to infect Rps1b-containing soybeans. However, loss of expression of the incoming Avr1b-1 gene was frequently observed in KI transformants, which resulted in these transformants readily infecting Rps1b soybeans. A similar variability in the expression levels of the incoming gene was observed with AVI- or mCherry-tagged Avr1b-1 constructs. Our results suggest that Avr1b-1 may be unusually susceptible to transcriptional variation.

As a destructive plant pathogen, Phytophthora infestans secretes diverse host-entering RxLR effectors to facilitate infection. One critical RxLR effector, PiAvr3b, not only induces effector-triggered immunity (ETI), which is associated with the potato resistance protein StR3b, but also suppresses pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). To date, the molecular basis underlying such dual activities remains unknown. Based on phylogenetic analysis of global P. infestans isolates, we found two PiAvr3b isoforms that differ by three amino acids. Despite this sequence variation, the two isoforms retain the same properties in activating the StR3b-mediated hypersensitive response (HR) and inhibiting necrosis induced by three PAMPs (PiNpp, PiINF1, and PsXeg1) and an RxLR effector (Pi10232). Using a combined mutagenesis approach, we found that the dual activities of PiAvr3b were tightly linked and determined by 88 amino acids at the C-terminus. We further determined that either the W60 or the E134 residue of PiAvr3b was essential for triggering StR3b-associated HR and inhibiting PiNpp-and Pi10232-associated necrosis, while the S99 residue partially contributed to PTI suppression. Additionally, nuclear localization of PiAvr3b was required to stimulate HR and suppress PTI, but not to inhibit Pi10232-associated cell death. Our study revealed that PiAvr3b suppresses the plant immune response at different subcellular locations and provides an example in which a single amino acid of an RxLR effector links ETI induction and cell death suppression.

Phytophthora capsici deploys effector proteins to manipulate host immunity and facilitate its colonization. However, the underlying mechanisms remain largely unclear. In this study, we demonstrated that a Sne-like (Snel) RxLR effector gene PcSnel4 is highly expressed at the early stages of P. capsici infection in Nicotiana benthamiana. Knocking out both alleles of PcSnel4 attenuated the virulence of P. capsici, while expression of PcSnel4 promoted its colonization in N. benthamiana. PcSnel4B could suppress the hypersensitive reaction (HR) induced by Avr3a-R3a and RESISTANCE TO PSEUDOMONAS SYRINGAE 2 (AtRPS2), but it did not suppress cell death elicited by Phytophthora infestin 1 (INF1) and Crinkler 4 (CRN4). COP9 signalosome 5 (CSN5) in N. benthamiana was identified as a host target of PcSnel4. Silencing NbCSN5 compromised the cell death induced by (AtRPS2). PcSnel4B impaired the interaction and co-localization of Cullin1 (CUL1) and CSN5 in vivo. Expression of AtCUL1 promoted the degradation of AtRPS2 and disrupted HR, while AtCSN5a stabilized AtRPS2 and promoted HR, regardless of the expression of AtCUL1. PcSnel4 counteracted the effect of AtCSN5 and enhanced the degradation of AtRPS2, resulting in HR suppression. This study deciphered the underlying mechanism of PcSnel4-mediated suppression of HR induced by AtRPS2.