The SsArl1 gene limits the virulence of Sclerotinia sclerotiorum

Deletion of the SsArl1 gene increased the virulence of Sclerotinia sclerotiorum in host plants, despite reducing hyphal growth. The result indicates an unusual function for this gene in the fungus. Instead of favoring infection, SsArl1 acts as a negative regulator of oxalic acid production, cellulase activity, and pathogen aggressiveness.

The research evaluated the function of SsArl1, a gene associated with the Arl family of small GTPases. These proteins participate in vesicular trafficking in eukaryotic cells. In the fungus Sclerotinia sclerotiorum, this system participates in the growth of hyphae and the secretion of factors related to pathogenicity.

Comparison with proteins

The researchers identified SsArl1 by comparing it to homologous proteins of Fusarium graminearum, Saccharomyces cerevisiae and humans. The analysis confirmed the gene's relationship with ADP-ribosylation factor-like 1 proteins. The study also pointed to a similarity with SsArf6, another protein from the Arf family already linked to vegetative growth and virulence in Sclerotinia sclerotiorum.

To investigate the gene's function, scientists produced a mutant with a deletion of SsArl1. They also generated a complemented lineage, in which the gene was reinserted. Comparisons involved the wild-type lineage, the ∆Ssarl1 mutant, and the ∆Ssarl1-C lineage.

The ∆Ssarl1 mutant showed reduced growth in potato-dextrose-agar medium. The difference appeared in evaluations performed after 24 and 48 hours. However, the deletion did not alter sclerotia formation or appressorium formation. The result indicates the gene's involvement in normal hyphal growth, but not in these two developmental processes.

Pathogenicity tests

In pathogenicity tests, the mutant caused larger lesions on leaves of Arabidopsis thaliana and Nicotiana benthamiana. The supplemented strain reduced infectivity compared to the mutant. This pattern supported the researchers' conclusion: SsArl1 reduces the virulence of Sclerotinia sclerotiorum.

The proposed explanation involves oxalic acid. This compound acts as a virulence factor in Sclerotinia sclerotiorum. The fungus uses oxalic acid to acidify tissues, interfere with plant defenses, and promote necrosis. In the study, the ∆Ssarl1 mutant showed greater acidification of the medium with bromophenol blue, even with smaller colonies.

Quantification by ultra-high performance liquid chromatography confirmed the increase in oxalic acid. Intracellular and extracellular levels were higher in the ∆Ssarl1 mutant than in the wild-type and supplemented strains. The expression of SsOAH1, a key gene in oxalic acid biosynthesis, was also increased in the mutant.

Greater activity

The researchers also observed increased secreted cellulase activity in the mutant. The combination of more oxalic acid and greater activity of cell wall-degrading enzymes helps explain the greater severity of the infection. The study proposes a model in which SsArl1 limits the expression of SsOAH1 and restricts oxalic acid secretion. With the deletion of the gene, this restriction disappears.

Environmental stresses

The research also evaluated responses to environmental stresses. The mutant showed greater sensitivity to ionic stress caused by sodium chloride and potassium chloride. In contrast, it did not demonstrate greater relevant sensitivity to Congo red or sodium dodecyl sulfate when the data were corrected for lower basal growth. Under non-ionic osmotic stress, with sorbitol and glucose, the mutant showed less relative growth inhibition.

The results differ from studies with other phytopathogenic fungi. In magnaporthe oryzae and Fusarium graminearum, the deletion of Arl1 homologs reduces growth and virulence. In Sclerotinia sclerotiorum, the deletion reduced growth but increased infection. The researchers point to possible functional divergence between Arl1 proteins in different pathogens.

Further information can be found at doi.org/10.3390/jof12060431