Horizontal transfer of a conserved npc-2 like effector gene in rust fungi that suppresses cell death in plants

ML/MD-2 is a conserved lipid/sterol-binding protein family having a role in sterol transfer and innate immunity in lower and higher eukaryotes. Here we report a genome-wide survey of this family, identifying 84 genes in 25 fungal and five oomycetes plant pathogen, having a different nutrition mode. All the fungal species were found to have varied numbers of family members, a distinctively substantial expansion of the ML gene family was observed in Rhizophagus irregularis (RI) with 33 genes. Our analysis also showed that NPC2 like proteins, a subfamily of ML domain superfamily, were not only restricted to animals and insect species but also present in plant fungal pathogens, including members of Clavicipitaceae, Pucciniacease, and Tremellaceae family. The phylogenetic analysis showed that these NPC2 like fungal proteins are more closely related to animals/insects than other fungal species. The molecular docking studies of these proteins with cholesterol and other derivatives indicate lipid-binding functional conservation across the animal and fungi kingdom. Further, the full length CDS of one of the npc2 like genes from Puccinia triticina (Pt5643) was PCR amplified and further characterized using various studies such as qRT-PCR, expression in onion epidermal cells, Nicotiana benthamiana for subcellular localization studies, yeast functional complementation, and expression studies. The mRNA abundance of Pt5643 was observed to be increased along with the infection progression and exhibits the highest expression at 5thday post-infection (dpi), suggesting its important role in the P. triticina infection cycle in wheat. The fluorescent confocal microscopy of transiently expressed YFP tagged Pt5643 in onion epidermal cells and N. benthamiana shows its location in cytoplasm and nucleus, indicating its involvement in the manipulation of host genes. The functional complementation of Pt5643 in npc2 mutant yeast showed its functional similarity to the eukaryotic npc2 gene. Further, the overexpression of Pt5643 also suppressed the BAX and H2O2 induced program cell death in N. benthamiana and yeast, respectively thus proving to be a novel horizontally transferred effector in rust fungal pathogens. Altogether the present study reports the novel function of fungal NPC2 like proteins playing a crucial role in host defense manipulation possibly through lipid binding/transport similar to animals.