Comparative Methods for Molecular Determination of Host-Specificity Factors in Plant-Pathogenic Fungi.

Many plant-pathogenic fungi are highly host-specific. In most cases, host-specific interactions evolved at the time of speciation of the respective host plants. However, host jumps have occurred quite frequently, and still today the greatest threat for the emergence of new fungal diseases is the acquisition of infection capability of a new host by an existing plant pathogen. Understanding the mechanisms underlying host-switching events requires knowledge of the factors determining host-specificity. In this review, we highlight molecular methods that use a comparative approach for the identification of host-specificity factors. These cover a wide range of experimental set-ups, such as characterization of the pathosystem, genotyping of host-specific strains, comparative genomics, transcriptomics and proteomics, as well as gene prediction and functional gene validation. The methods are described and evaluated in view of their success in the identification of host-specificity factors and the understanding of their functional mechanisms. In addition, potential methods for the future identification of host-specificity factors are discussed.

Biotrophy-specific downregulation of siderophore biosynthesis in Colletotrichum graminicola is required for modulation of immune responses of maize.

The hemibiotrophic maize pathogen Colletotrichum graminicola synthesizes one intracellular and three secreted siderophores. eGFP fusions with the key siderophore biosynthesis gene, SID1, encoding l-ornithine-N(5) -monooxygenase, suggested that siderophore biosynthesis is rigorously downregulated specifically during biotrophic development. In order to investigate the role of siderophores during vegetative development and pathogenesis, SID1, which is required for synthesis of all siderophores, and the non-ribosomal peptide synthetase gene NPS6, synthesizing secreted siderophores, were deleted. Mutant analyses revealed that siderophores are required for vegetative growth under iron-limiting conditions, conidiation, ROS tolerance, and cell wall integrity. Δsid1 and Δnps6 mutants were hampered in formation of melanized appressoria and impaired in virulence. In agreement with biotrophy-specific downregulation of siderophore biosynthesis, Δsid1 and Δnps6 strains were not affected in biotrophic development, but spread of necrotrophic hyphae was reduced. To address the question why siderophore biosynthesis is specifically downregulated in biotrophic hyphae, maize leaves were infiltrated with siderophores. Siderophore infiltration alone did not induce defence responses, but formation of biotrophic hyphae in siderophore-infiltrated leaves caused dramatically increased ROS formation and transcriptional activation of genes encoding defence-related peroxidases and PR proteins. These data suggest that fungal siderophores modulate the plant immune system.

An adequate Fe nutritional status of maize suppresses infection and biotrophic growth of Colletotrichum graminicola.

Iron (Fe) is an essential element for plant pathogens as well as for their host plants. As Fe plays a central role in pathogen virulence, most plants have evolved Fe-withholding strategies to reduce Fe availability to pathogens. On the other hand, plants need Fe for an oxidative burst in their basal defense response against pathogens. To investigate how the plant Fe nutritional status affects plant tolerance to a hemibiotrophic fungal pathogen, we employed the maize-Colletotrichum graminicola pathosystem. Fungal infection progressed rapidly via biotrophic to necrotrophic growth in Fe-deficient leaves, while an adequate Fe nutritional status suppressed the formation of infection structures of C. graminicola already during the early biotrophic growth phase. As indicated by Prussian blue and 3,3'-diaminobenzidine (DAB) staining, the retarding effect of an adequate Fe nutritional status on fungal development coincided temporally and spatially with the recruitment of Fe to infection sites and a local production of H2 O2 . A similar coincidence between local Fe and H2 O2 accumulation was found in a parallel approach employing C. graminicola mutants affected in Fe acquisition and differing in virulence. These results indicate that an adequate Fe nutritional status delays and partially suppresses the fungal infection process and the biotrophic growth phase of C. graminicola, most likely via the recruitment of free Fe to the fungal infection site for a timely oxidative burst.

Infection structure-specific reductive iron assimilation is required for cell wall integrity and full virulence of the maize pathogen Colletotrichum graminicola.

Ferroxidases are essential components of the high-affinity reductive iron assimilation pathway in fungi. Two ferroxidase genes, FET3-1 and FET3-2, have been identified in the genome of the maize anthracnose fungus Colletotrichum graminicola. Complementation of growth defects of the ferroxidase-deficient Saccharomyces cerevisiae strain Δfet3fet4 showed that both Fet3-1 and Fet3-2 of C. graminicola represent functional ferroxidases. Expression of enhanced green fluorescent protein fusions in yeast and C. graminicola indicated that both ferroxidase proteins localize to the plasma membrane. Transcript abundance of FET3-1 increased dramatically under iron-limiting conditions but those of FET3-2 were hardly detectable. Δfet3-1 and Δfet3-2 single as well as Δfet3-1/2 double-deletion strains were generated. Under iron-sufficient or deficient conditions, vegetative growth rates of these strains did not significantly differ from that of the wild type but Δfet3-1 and Δfet3-1/2 strains showed increased sensitivity to reactive oxygen species. Furthermore, under iron-limiting conditions, appressoria of Δfet3-1 and Δfet3-1/2 strains showed significantly reduced transcript abundance of a class V chitin synthase and exhibited severe cell wall defects. Infection assays on intact and wounded maize leaves, quantitative data of infection structure differentiation, and infection stage-specific expression of FET3-1 showed that reductive iron assimilation is required for appressorial penetration, biotrophic development, and full virulence.