A Phytophthora infestans CRN1-derived small RNA is predicted to target the potato immune regulator EDS1

The late blight pathogen, Phytophthora infestans (Pi), causes severe damage to plants in the Solanaceae family. Although knowledge regarding the P. infestans-mediated manipulation of critical components in the plant defense system is growing, many questions remain unanswered. Herein, we aimed to examine the role of Argonaute 1 (AGO1) associated small RNAs in this interaction. Of particular interest was the early communication between the host and the pathogen. To visualize the cellular dynamics underlying potential cross-kingdom RNA trafficking, we first examined the localization and accumulation patterns of plant extracellular vesicles (EVs) and multivesicular bodies (MVBs) using a handful of markers. MVBs were present not only at the plant plasma membrane but also in the germ tube of the invading pathogen. The enrichment of MVBs decreased as the infection process proceeded. At 3.0 days post-inoculation, co-localization between AGO1 from P. infestans and StARA6 was not seen even at the swollen tip of the germ tube. Three Crinkler effector genes encoding small RNAs were found after coimmunoprecipitation, sequencing and extensive bioinformatic analysis. PiCRN1 caused more severe disease compared with PiCRN3, which carries a typical Crinkler (CRN) LFLAK domain. This difference may result from activation of a CRN1-derived siRNA predicted to target the enhanced disease susceptibility 1 (EDS1) gene in potato. To examine whether the observed phenotypic effects can be attributed to any EV cargo from the potato host, we set up a procedure to isolate EVs from P. infestans-infected potato leaves. However, the tiny EV yield obtained during the early infection phase prevented us from such analysis. The understanding of effector cell trafficking and small RNA reprogramming of host plant genes remain elusive in this pathosystem.