Trifluralin herbicide-induced resistance of melon to fusarium wilt involves expression of stress- and defence-related genes

To identify genes involved in trifluralin herbicide-induced resistance of melon to Fusarium oxysporum f. sp. melonis , suppression subtractive hybridization (SSH) and cDNA-amplified fragment-length polymorphism (cDNA-AFLP) were used. A total of 123 clones—60 of which have never been isolated from melon—were isolated, sequenced and annotated. A significant proportion (35%) of the total 123 clones exhibited similarity to genes that have been formerly described as stress- or defence-related. Thirty-two selected clones were subjected to a detailed expression analysis, one-third of which were found to be up-regulated in response to trifluralin treatment and/or fusarium inoculation. The putative roles of seven of these clones in stress are discussed. Furthermore, the expression of four stress-related and up-regulated genes was enhanced when the plants were subjected to salinity stress, suggesting that trifluralin induces a general stress response which protects the plant against fusarium wilt.     

The global nitrogen regulator, FNR1, regulates fungal nutrition-genes and fitness during Fusarium oxysporum pathogenesis

Fusarium oxysporum is a soil-borne pathogen that infects plants through the roots and uses the vascular system for host ingress. Specialized for this route of infection, F. oxysporum is able to adapt to the scarce nutrient environment in the xylem vessels. Here we report the cloning of the F. oxysporum global nitrogen regulator, Fnr1 , and show that it is one of the determinants for fungal fitness during in planta growth. The Fnr1 gene has a single conserved GATA-type zinc finger domain and is 96% and 48% identical to AREA-GF from Gibberella fujikuroi , and NIT2 from Neurospora crassa , respectively. Fnr1 cDNA, expressed under a constitutive promoter, was able to complement functionally an N. crassa nit-2 ^RIP mutant, restoring the ability of the mutant to utilize nitrate. Fnr1 disruption mutants showed high tolerance to chlorate and reduced ability to utilize several secondary nitrogen sources such as amino acids, hypoxanthine and uric acid, whereas growth on favourable nitrogen sources was not affected. Fnr1 disruption also abolished in vitro expression of nutrition genes , normally induced during the early phase of infection. In an infection assay on tomato seedlings, infection rate of disruption mutants was significantly delayed in comparison with the parental strain. Our results indicate that FNR1 mediates adaptation to nitrogen-poor conditions in planta through the regulation of secondary nitrogen acquisition, and as such acts as a determinant for fungal fitness during infection.     

Efficient gene replacement and direct hyphal transformation in Sclerotinia sclerotiorum

Homologous recombination is required for gene-targeted procedures such as gene disruption and gene replacement. Ku80 is part of the non-homologous end-joining DNA repair mechanism in many organisms. We identified and disrupted the Ku80 homologue in Sclerotinia sclerotiorum and generated heterokaryon mutants enriched with Ku80 -deficient nuclei ( ssku80 ). Sclerotial formation and pathogenicity of ssku80 mutants were normal on tomato fruits. The frequencies of homologous recombination in these strains were much higher than those of the wild type when transformed with a cna1 (encoding calcineurin) replacement construct. We coupled the increase in homologous recombination with a direct BIM-LAB-mediated transformation procedure, which utilizes compressed air to assist the transforming DNA in penetrating fungal hyphae of S. sclerotiorum . We found this method to be efficient and reproducible, and it did not alter the fitness of the mutants. We also demonstrated the first case of direct transformation of sclerotia. Nourseothricin was introduced as a selectable marker in S. sclerotiorum . The tools and procedures described will improve our ability to study gene function in S. sclerotiorum and are most likely to be adaptable for use in other plant pathogens.