Small RNAs and cysteine-rich proteins - novel molecular weapons of oomycete plant pathogens

Oomycetes comprise infamous plant pathogens that jeopardize global food resources. Effector proteins promote infections of pathogens and are consequently also found in large numbers in the genome of oomycetes. More recently, it was discovered that in fungi also small RNAs (sRNAs) can act as effectors by silencing host immunity genes by cross-kingdom RNA interference (RNAi). The effector diversity results in a highly complex, multilayered host pathogen cross-talk, whereas oomycete effector research has been mainly focused on a single effector class: the RxLR effectors. Consequently, I wanted to investigate two understudied oomycete effector classes: sRNAs and small secreted non-RxLR cysteine-rich (CR) proteins. I adapted an immunopurification-based method for high-throughput sequencing of pathogen sRNAs that were associated with the host ARGONAUTE (AGO) protein forming the RNA-induced silencing complex (RISC). From our first insights into the putative sRNA effectors of H. arabidopsidis I selected candidates that revealed target transcript repression. I designed a novel in situ reporter that demonstrated both pathogen sRNA translocation into plants and efficient host target silencing. Thereby, I directly visualized the spatial dimension of cross-kingdom RNAi in the host tissue. I validated the crucial contribution of sRNAs to virulence by scavenging three of them with a plant encoded sRNA target mimic array. Hereby, I introduced H. arabidopsidis as a complementary cross-kingdom RNAi model to B. cinerea. I suggest that pathogen sRNA effectors are not only a widespread virulence mechanism, but also that comparative research can enlighten the evolutionary forces that shape sRNA arsenals in pathogens with distinct lifestyles and host ranges. As H. arabidopsidis remains inaccessible to classical reverse genetics, me and my colleagues established host-induced gene silencing (HIGS) in Arabidopsis as a tool to knock down and study non-RxLR HaCR1 protein effector function. Isolated Arabidopsis HaCR1RNAi lines displayed prominent host cell death upon H. arabidopsidis infection suggesting that HaCR1 inhibits induced plant cell death to promote infection. HaCR1 seemed to reside in the plant apoplast and its activity was strictly dependent on its signal peptide. We found that HaCR1 inhibited plant extracellular protease activity, suggesting that HaCR1 might interfere with plant defensive proteases and protease-dependent programmed cell death (PCD), providing for the first time, insights into the molecular function of a H. arabidopsidis apoplastic effector. Taken together, I provide new insights into the role of sRNAs and cysteine-rich protein effectors for H. arabidopsidis host infection. Completing the picture of the pathogen virulence arsenal poses an important prerequisite towards more effective pathogen control