Genomic insights into the pathogenicity and host adaptation in species of the Leptographium wageneri complex

Abstract

Fungal vascular wilt pathogens cause destructive diseases in many agriculturally important crop and tree species. Many of these pathogens are soil-borne and enter their hosts via pre-existing structures, or with the aid of insect vectors. However, there is remarkably little knowledge available regarding the molecular mechanisms of infection and pathogenicity in these fungi. Species in the Leptographium wageneri complex cause black stain root disease of conifers. This disease is characterised by black staining of the lower stems, roots, and tracheids, leading to wilt and tree death. There are three host specific species in the complex: L. wageneri, L. pseudotsugae and L. ponderosum. The genetic mechanisms underlying their pathogenicity and host adaptation have not been identified. To better understand potential pathogenicity associated factors, comparative genomic analysis was performed by comparing the genomes of species in the L. wageneri complex with those from related non-pathogenic species that reside in the same genus as well as genomes of pathogenic and non-pathogenic species in the Ophiostomataceae. The in vitro and in planta expression of an identified laccase gene was investigated to reveal its role in pathogenicity. Furthermore, laccasedeletion mutants were generated using the CRISPR-Cas9 gene editing system, followed by a pathogenicity trial on Pinus patula seedlings. Finally, a genome-wide approach was used to identify genomic regions under selection that might explain the host specialization in species of the L. wageneri complex. Genome sequences were generated for the three species in the L. wageneri complex and the nonpathogenic L. douglasii. The results revealed that species in the L. wageneri complex have larger genomes, higher gene numbers and a higher genome-wide content of transposable elements. Overall, both pathogenic and non-pathogenic species in the Ophiostomataceae have similar numbers of pathogenicity associated factors, such as secondary metabolite clusters, CAZymes, and effectors. Phylogenetic analyses indicated that the laccase gene has been horizontally acquired by species of the L. wageneri complex and L. douglasii. Expression analysis revealed that the laccase gene is up regulated in planta. The pathogenicity trial conducted with laccase-deletion mutants confirmed the essential role of the laccase gene in pathogenicity. Whole-genome SNP data were used to investigate the evolutionary relationships and genetic patterns of diversification between species of the L. wageneri complex. Structure analysis revealed three distinct clusters, each representing a species in the complex. Evolutionary analyses indicated that L. wageneri is more closely related to L. pseudotsugae, than to L. ponderosum. Selection analysis revealed several genomic regions underwent positive selection, that could explain their differentiation and host associations. Finally, I have discussed how genes within these genomic regions that encode for a heterochromatin incompatibility protein, a protein kinase and a Multi-drug transporter protein could underly the diversification and host specification in the species of the L. wageneri complex.