Characterization of the Largest Effector Gene Cluster of Ustilago maydis

Abstract

In the genome of the biotrophic plant pathogen Ustilago maydis, many of the genes coding for secreted protein effectors modulating virulence are arranged in gene clusters. The vast majority of these genes encode novel proteins whose expression is coupled to plant colonization. The largest of these gene clusters, cluster 19A, encodes 24 secreted effectors. Deletion of the entire cluster results in severe attenuation of virulence. Here we present the functional analysis of this genomic region. We show that a 19A deletion mutant behaves like an endophyte, i.e. is still able to colonize plants and complete the infection cycle. However, tumors, the most conspicuous symptoms of maize smut disease, are only rarely formed and fungal biomass in infected tissue is significantly reduced. The generation and analysis of strains carrying sub-deletions identified several genes significantly contributing to tumor formation after seedling infection. Another of the effectors could be linked specifically to anthocyanin induction in the infected tissue. As the individual contributions of these genes to tumor formation were small, we studied the response of maize plants to the whole cluster mutant as well as to several individual mutants by array analysis. This revealed distinct plant responses, demonstrating that the respective effectors have discrete plant targets. We propose that the analysis of plant responses to effector mutant strains that lack a strong virulence phenotype may be a general way to visualize differences in effector function.

Author Summary

In this study, we provide the first step to the functional analysis of the largest gene cluster in the Ustilago maydis genome encoding 24 secreted effectors. While the deletion of the entire cluster dramatically affected tumor formation and abolished anthocyanin induction, only one of the genes had a large contribution to tumor formation, while another effector gene was primarily responsible for the anthocyanin induction. Unexpectedly, the cluster mutant could still colonize plants and complete the life cycle, i.e. behaves like an endophyte. Despite only small contributions to tumor formation, individual effector mutants caused distinct plant responses, suggesting that they affect discrete plant processes. On these grounds we are proposing to use plant responses as a general readout to assess and compare effector gene function.