Characterization and functional roles of the flavohemoglobin genes of the plant pathogen Fusarium oxysporum

Eduardo Argotti Jose M. Diaz-Minguez Ernesto P. Benito
CIALE (Instituto Hispano-Luso de Investigaciones Agrarias), Departamento de Microbiología y Genética, Universidad de Salamanca, Villamayor, Spain

Nitric oxide (NO) is a highy reactive molecule playing important roles in essential developmental processes and stress responses. Phytopathogenic fungi use NO as a signalling molecule to modulate development but, concurrently, they have to counteract the plant defensive responses primed by NO. Fungi have evolved several mechanisms against nitrosative stress, among them the detoxification of NO radicals by the enzymes flavohemoglobins is a prominent one.

Genome analysis of Fusarium oxysporum showed that this fungus present four genes encoding flavohemoglobins. FHG1, FHG2, and FHG3 encode fungal cytoplasmatic flavohemoglobins, while FHG4 encodes a flavohemoglobin of a probable bacterial origin, as indicated by the clustering in phylogenetic analyses and the putative mitochondrial localization. Gene expression analyses performed with F. oxysporum f. sp. phaseoli strains grown in several nitrogen sources and exposed to NO donors, showed that FHG1 and FHG2 increased their transcript levels during spore germination. FHG3 showed a complex regulation while FHG4 did not show any transcriptional response in the assayed conditions. Functional analysis of mutants deleted in the FHG2 gene and silenced in the FHG1 gene in both strains, confirmed that FHG1 plays the major role and FHG2 a minor role in NO detoxification, which confers WV and HV virulent strains resistance to nitrosative stress and prevents the delay in spore germination produced by high levels of NO.

Transcript levels of the four genes were also determined in common bean plants (Phaseolus vulgaris L.) inoculated with WV and HV strains. The FHG1 transcript acumulation increased after infection and reached maximum levels by the first week of colonization, while the three other genes did not show changes in expression. However, inoculation assays performed either with single mutants deleted in FHG2 or double mutants deleted in FHG2 and silenced in FHG1, did not evidence that these genes are pathogenicity or virulence factors.