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1.  Characterization of the Largest Effector Gene Cluster of Ustilago maydis 
PLoS Pathogens  2014;10(7):e1003866.
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.
PMCID: PMC4081774  PMID: 24992561
2.  A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesis in maize 
eLife  2014;3:e01355.
The biotrophic fungus Ustilago maydis causes smut disease in maize with characteristic tumor formation and anthocyanin induction. Here, we show that anthocyanin biosynthesis is induced by the virulence promoting secreted effector protein Tin2. Tin2 protein functions inside plant cells where it interacts with maize protein kinase ZmTTK1. Tin2 masks a ubiquitin–proteasome degradation motif in ZmTTK1, thus stabilizing the active kinase. Active ZmTTK1 controls activation of genes in the anthocyanin biosynthesis pathway. Without Tin2, enhanced lignin biosynthesis is observed in infected tissue and vascular bundles show strong lignification. This is presumably limiting access of fungal hyphae to nutrients needed for massive proliferation. Consistent with this assertion, we observe that maize brown midrib mutants affected in lignin biosynthesis are hypersensitive to U. maydis infection. We speculate that Tin2 rewires metabolites into the anthocyanin pathway to lower their availability for other defense responses.
eLife digest
The production of agricultural crop plants is severely hindered by bacteria, viruses, and fungi that have developed their own strategies to colonize these plants and obtain nutrients from them. Some pathogens kill the plants they colonize, but ‘biotrophic pathogens’ employ sophisticated strategies to manipulate the host plant without killing it.
During the past decade it has been recognized that the interactions between plants and biotrophic pathogens are largely governed by effector proteins—which are typically secreted by the pathogen after it makes contact with the host. These effector proteins can either stay in the space between the plant cells and pathogen cells, or actually enter inside the plant cells.
The fungus Ustilago maydis is one such biotrophic pathogen that colonizes maize plants and causes a disease called corn smut. Hallmarks of this infection are the formation of large plant tumors and the production of a red pigment, called anthocyanin, in infected plant tissues.
Now, Tanaka et al. reveal that an effector called Tin2, which is secreted by the corn smut fungus, causes the production of this anthocyanin pigment. Tin2 moves inside plant cells, where it blocks the breakdown of a protein-modifying enzyme that is necessary to ‘switch on’ the production of anthocyanin. When a mutant fungus that lacks Tin2 infects a maize plant, no anthocyanin is induced and the pathogen fails to reach the vascular tissue, where it would normally get most of its nutrients. Tanaka et al. revealed that, in these infections, this vascular tissue was strongly reinforced with a compound, called lignin, suggesting that the plant fortifies these cell walls to block access by the mutant fungus.
Since the building blocks for making lignin are also required for making anthocyanins, Tanaka et al. suggest a model whereby Tin2 compromises the ability of plants to protect themselves by diverting resources away from making lignin. In line with this speculation, the corn smut fungus was shown to cause stronger disease symptoms in maize plants with mutations that prevent them from producing lignin.
The Tin2 effector of the corn smut fungus appears to target a critical protein in maize that can shift the balance of the plant’s metabolic pathways in favor of the pathogen. Further, since anthocyanin production is also observed after infections of plants with other microbes, these findings may have uncovered a microbial strategy to enhance virulence that is also employed by other plant pathogens.
PMCID: PMC3904489  PMID: 24473076
Ustilago maydis; smut fungus; plant pathogen; Other
3.  HvCEBiP, a gene homologous to rice chitin receptor CEBiP, contributes to basal resistance of barley to Magnaporthe oryzae 
BMC Plant Biology  2010;10:288.
Rice CEBiP recognizes chitin oligosaccharides on the fungal cell surface or released into the plant apoplast, leading to the expression of plant disease resistance against fungal infection. However, it has not yet been reported whether CEBiP is actually required for restricting the growth of fungal pathogens. Here we evaluated the involvement of a putative chitin receptor gene in the basal resistance of barley to the ssd1 mutant of Magnaporthe oryzae, which induces multiple host defense responses.
The mossd1 mutant showed attenuated pathogenicity on barley and appressorial penetration was restricted by the formation of callose papillae at attempted entry sites. When conidial suspensions of mossd1 mutant were spotted onto the leaves of HvCEBiP-silenced plants, small brown necrotic flecks or blast lesions were produced but these lesions did not expand beyond the inoculation site. Wild-type M. oryzae also produced slightly more severe symptoms on the leaves of HvCEBiP-silenced plants. Cytological observation revealed that these lesions resulted from appressorium-mediated penetration into plant epidermal cells.
These results suggest that HvCEBiP is involved in basal resistance against appressorium-mediated infection and that basal resistance might be triggered by the recognition of chitin oligosaccharides derived from M. oryzae.
PMCID: PMC3020183  PMID: 21190588

Results 1-3 (3)