Multiomic approaches reveal novel lineage-specific effectors in the potato and tomato early blight pathogen Alternaria solani

Abstract The effectome of the necrotrophic fungal pathogen, Alternaria solani, was determined using multiomics. In total, 238 effector candidates were predicted from the A. solani genome, and apoplastic effectors constitute most of the total candidate effector proteins (AsCEPs). Comparative genomics revealed two main groups of AsCEPs: lineage-specific and conserved effectors. RNA-Seq analysis revealed that the most highly expressed genes encoding AsCEPs were enriched with lineage-specific forms. Two lineage-specific effector genes, AsCEP19 and AsCEP20, were found to form a ‘head-to-head’ gene pair located near an AT-rich region on the chromosome. To date, AsCEP19 and AsCEP20 have been found only in a few fungal species. Phylogenetic inference revealed that AsCEP19 and AsCEP20 were likely acquired by the common ancestor of A. solani and A. tomatophila via horizontal gene transfer, probably mediated by long terminal repeat retrotransposon. RT-qPCR analysis showed that AsCEP19 and AsCEP20 are tightly coexpressed in a host-specific manner and that they are upregulated at advanced stages of A. solani infection only in solanaceous hosts. Transient expression of AsCEP19 and AsCEP20 in Nicotiana benthamiana plants showed that these effectors could promote Phytophthora infestans infection. AsCEP19 and AsCEP20 were required for the full virulence of A. solani on host potato, because deletion of this gene pair significantly reduced the size of necrotic lesions on potato leaves. Transient expression of AsCEP20 could elicit plant cell death depending on the presence of its signal peptide, indicating that AsCEP20 is a necrosis-inducing apoplastic effector with the mature form localized specifically in chloroplasts. Our work provides a better understanding of the function and evolution of necrotrophic fungal effectors, and helps explain the high aggressiveness of A. solani against solanaceous crops.