Identification and Characterization of Phytophthora Effectors with RNA Silencing Suppression Activity

Phytophthora are filamentous eukaryotes that contain many important pathogens of plants. These destructive pathogens bring economic losses of billions of dollars per year. My thesis research aims to understand the molecular mechanisms involved in Phytophthora pathogenesis. In particular, I am interested in understanding how Phytophthora pathogens defeat plant immunity. Existing evidence have revealed the never-ending molecular arms race between pathogens and their hosts. Recent research discovered that small RNAs play an important regulatory role in plant defense. However, Phytophthora pathogens encode effectors with RNA silencing suppression activities to manipulate small RNA-mediated plant defense. In this thesis, I characterized the host target of the Phytophthora RNA silencing suppressor 2 (PSR2) and identified additional RNA silencing suppressors from Phytophthora species.In chapter I, I characterized the host target of PSR2. PSR2 was previously identified from Phytophthora sojae and specifically affects secondary small interfering RNA (siRNA) accumulation in Arabidopsis. Using genetics and molecular biology approaches, I found that PSR2 interacts with the double-stranded RNA binding protein 4 (DRB4) in plant cells. DRB4 has a known function in secondary siRNA biogenesis by partnering with the endonuclease Dicer-like protein 4 (DCL4), which processes long double-stranded RNA (dsRNA) precursors into siRNAs. DRB4 contains two double-stranded RNA binding motifs (dsRBMs) in the N terminus, which are required for the interaction with PSR2. In addition, I also determined that the N-terminus of PSR2 is necessary and sufficient to interact with DRB4. Importantly, this N-terminal region of PSR2 is also necessary and sufficient for the virulence activity of PSR2. These results indicate DRB4 is likely a major virulence target of PSR2 to promote Phytophthora infection. In chapter II, I identified additional effectors that suppress RNA silencing in plants. Previous research on Phytophthora has focused on cytoplasmic effectors that contain the “RXLR” motif. In order to examine the prevalence of RNA silencing suppressors in Phytophthora, I screened a collection of the Crinkler effectors, which contain a different host-targeting motif. The CRN effectors are also unique in that: 1) they are usually constitutively expressed; 2) they are nuclear-localized. I found two CRNs from Phytophthora capsici as potential RNA silencing suppressors. Both effectors are widely spread in other Phytophthora species. In particular, CRN36_259 was able to reduce the accumulation of siRNAs in Nicotiana benthamiana while CRN32_283 suppressed RNA silencing without affecting siRNA levels. Interestingly, the nuclear localization of CRN36_259 is required for its RNA silencing suppression activity. In addition, the CRN36_259 knockout mutant of P. capsici exhibited altered developmental phenotype and virulence activity. These results suggest that CRN36_259 regulates Phytophthora development and virulence, possibly through its function as a suppressor of small RNAs.This thesis provides fundamental knowledge about Phytophthora pathogenesis and sets the foundation to develop resistance against the destructive Phytophthora diseases