Identification and partial characterisation of a putative ribonuclease effector in the pitch canker pathogen Fusarium circinatum.

Schultze, S. P., Visser, E. A., Naidoo, S.*

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0028, South Africa.

The presence of Fusarium circinatum poses a threat to pine plantations and nurseries worldwide. One highly susceptible species to F. circinatum infection is Pinus patula, one of the most used softwood species in South Africa. Contemporary control strategies for F. circinatum are insufficient. It is therefore imperative to investigate and elucidate the pathogen-host molecular dialog to mitigate the destructive capacity of this pathogen. Previous dual RNA-seq data of F. circinatum-inoculated resistant and susceptible pine seedlings at 3- and 7 days post inoculation (dpi) was used to identify putative core F. circinatum virulence factors. RT-qPCR of inoculated Pinus tecunumanii (low elevation provenance) and Pinus patula seedlings, from a new trial, coupled with an Agrobacterium-mediated transient transformation assay was used to partially characterise a novel F. circinatum putative ribonuclease effector. Eight putative core virulence factors were identified to be involved in this pathogen-host interaction. These eight genes included extracellular cell wall glucanase, transglycosylase, SED-1 abundant glycoprotein, Fusarium osmotin resistance, GzOB030 transcription factor, trehalose-6-phosphate, arabinogalactan endo-1,4-beta-galactosidase, and a guanyl-specific ribonuclease. Expression of all eight genes was confirmed in planta at 3 and 7 dpi. One candidate, with high similarity to a guanyl-specific ribonuclease (Fg12) in Fusarium graminearum predicted to induce host cell death through total RNA degradation, was selected for further investigation. The F. circinatum putative ribonuclease protein (FcPRP) was found to be similar to Fg12 through protein domain, structural, and phylogenetic analyses. Additionally, FcPRP was one of the highest expressed genes in the pathogen-host interaction. FcPRP was further characterised using transient expression, which confirmed the induction of a hypersensitive response through cell death in Nicotiana benthamiana. This research contributes to the contemporary understanding of the F. circinatum arsenal, deployed to efficiently colonize and cause disease in host Pinus seedlings, identifying the ribonuclease FcPRP as a key player in fungal virulence and a promising target for novel pitch canker disease control. The findings of this study may be used in future work as a basis for developing novel strategies to control F. circinatum.

Keywords: Fusarium circinatum, Pinus tecunumanii, Pinus patula, virulence factors, effector characterisation