Hce2 domain-containing effectors contribute to the full virulence of Valsa mali in a redundant manner.

Valsa mali is the causal agent of apple Valsa canker, a destructive disease in East Asia. Effector proteins play important roles in the virulence of phytopathogenic fungi, and we identified five Hce2 domain-containing effectors (VmHEP1, VmHEP2, VmHEP3, VmHEP4 and VmHEP5) from the V. mali genome. Amongst these, VmHEP1 and VmHEP2 were found to be up-regulated during the early infection stage and VmHEP1 was also identified as a cell death inducer through its transient expression in Nicotiana benthamiana. Although the deletion of each single VmHEP gene did not lead to a reduction in virulence, the double-deletion of VmHEP1 and VmHEP2 notably attenuated V. mali virulence in both apple twigs and leaves. An evolutionary analysis revealed that VmHEP1 and VmHEP2 are two paralogues, under purifying selection. VmHEP1 and VmHEP2 are located next to each other on chromosome 11 as tandem genes with only a 604 bp physical distance. Interestingly, the deletion of VmHEP1 promoted the expression of VmHEP2 and, vice versa, the deletion of VmHEP2 promoted the expression of VmHEP1. The present results provide insights into the functions of Hce2 domain-containing effectors acting as virulence factors of V. mali, and provide a new perspective regarding the contribution of tandem genes to the virulence of phytopathogenic fungi.