Ofir Degani

Distinct and Combined Roles of the MAP Kinases of Cochliobolus heterostrophus in Virulence and Stress Responses

Pathogenicity mitogen-activated protein kinases (MAPKs), related to yeast FUS3/KSS1, are essential for virulence in fungi, including Cochliobolus heterostrophus, a necrotrophic pathogen causing Southern corn leaf blight. We compared the phenotypes of mutants in three MAPK genes: HOG1, MPS1, and CHK1. The chk1 and mps1 mutants show autolytic appearance, light pigmentation, and dramatic reduction in virulence and conidiation. Similarity of mps1 and chk1 mutants is reflected by coregulation by these two MAPKs of several genes. Unlike chk1, mps1 mutants are female-fertile and form normal-looking appressoria. HOG1 mediates resistance to hyperosmotic and, to a lesser extent, oxidative stress, and is required for stress upregulation of glycerol-3-phosphate phosphatase, transaldolase, and a monosaccharide transporter. Hog1, but not Mps1 or Chk1, was rapidly phosphorylated in response to increased osmolarity. The hog1 mutants have smaller appressoria and cause decreased disease symptoms on maize leaves. Surprisingly, loss of MPS1 in a wild-type or hog1 background improved resistance to some stresses. All three MAPKs contribute to the regulation of central developmental functions under normal and stress conditions, and full virulence cannot be achieved without appropriate input from all three pathways.

Host physiology and pathogenic variation of Cochliobolus heterostrophus strains with mutations in the G protein alpha subunit, CGA1

ABSTRACT Conserved eukaryotic signaling proteins participate in development and disease in plant-pathogenic fungi. Strains with mutations in CGA1, a heterotrimeric G protein G alpha subunit gene of the maize pathogen Cochliobolus heterostrophus, are defective in several developmental pathways. Conidia from CGA1 mutants germinate as abnormal, straight-growing germ tubes that form few appressoria, and the mutants are female sterile. Nevertheless, these mutants can cause normal lesions on plants, unlike other filamentous fungal plant pathogens in which functional homologues of CGA1 are required for full virulence. Δcga1 mutants of C. heterostrophus were less infective of several maize varieties under most conditions, but not all, as virulence was nearly normal on detached leaves. This difference could be related to the rapid senescence of detached leaves, since delaying senescence with cytokinin also had differential effects on the virulence of the wild type and the Δcga1 mutant. In particular, detached leaves may provide a more readily available nutrient source than attached leaves. Decreased fitness of Δcga1 as a pathogen may reflect conditions under which full virulence requires signal transduction through CGA1-mediated pathways. The virulence of these signal transduction mutants is thus affected differentially by the physiological state of the host.

CHEMICAL CONTROL OF MAIZE LATE WILT IN THE FIELD

Late wilt, a severe vascular disease of maize caused by the fungus Harpophora maydis, is characterized by relatively rapid wilting of maize plants before tasseling and until shortly before maturity. In Israel, the disease has become a major problem in recent years. The pathogen is currently controlled using cultivars of maize having reduced sensitivity. In an earlier work, we modified a molecular method for use as a diagnostic tool to evaluate disease progression in field-infested plants and showed that several fungicides suppressed H. maydis in vitro. Here, we examine the effect of different fungicides on disease progression in a contaminated maize field in the spring and summer of 2009 and 2010. The field was watered using a drip irrigation line for each row and the fungicides were injected directly into the drip line. One of the four fungicides tested, azoxystrobin, was highly effective compared with the control, inhibited the development of wilt symptoms and recovered cob yield by 100%. Although this is the first success in preventing disease symptoms in infested fields in Israel, the azoxystrobin treatment did not reduce the amounts of pathogen DNA in host tissues or delay its spread. Attempts to reduce concentrations of this fungicide or to apply it by spraying were less effective than the triple full dosage treatment. The presence of the pathogen in the host tissues of the successfully treated plants and its ability to undergo pathogenic variations are increasing the risk of pathogen resistance and the urgent need to develop new ways of controlling late wilt.

Cochliobolus heterostrophus T-toxin Gene Expression Modulation via G Protein and MAPK Pathways

Degani O. (2015): Cochliobolus heterostrophus T-toxin gene expression modulation via G protein and

Mediation of fludioxonil fungicide activity and resistance through Cochliobolus heterostrophus G-protein and MAPK signaling pathways

The heterotrimeric G proteins and mitogen-activated protein kinases (MAPKs) conserved signaling pathways are involved in the development, reproduction and pathogenicity in filamentous fungi. The two-component histidine kinase, known also as the HOG MAPK pathway, regulates a similar complex set of responses and is known to mediate the phenylpyrrole fludioxonil fungicide response in fungi. We used Cochliobolus heterostrophus mutant strains deficient in G-protein α (cga1) and/or β (cgb1) subunits or MAPK (chk1, mps1 and hog1) to uncover their role in the mediation of this fungicide’s activity and resistance. The results revealed complex interactions between the G-protein subunits and the MAPK in response to osmotic/ionic and fludioxonil stresses. Under normal conditions, the Hog1 pathway restricts glycerol accumulation since its disruption leads to hyperosmosensitivity and very high cellular glycerol accumulation. The hog1 mutants were also relatively resistance to fludioxonil. Moreover, our results suggest that cgb1, chk1 and mps1 are also weak repressors of this response since mutation in these genes caused relatively high elevation in glycerol levels in the cells. Supporting this is the finding that these three strains exhibit resistance to KCl stress. In contrast, the cga1 strain has only moderate levels of cellular glycerol (higher than those of the wild type, but lower than those of the other mutants) that are little affected by KCl or fludioxonil stress. Indeed, these mutants are highly sensitive to KCl stress. This suggests that Cga1 is a moderate repressor of cellular glycerol under normal conditions and an enhancer of glycerol accumulation under osmotic/ionic stress conditions. Together these findings reveals that the sensitivity to fludioxonil is not only positively controlled by the Hog1 pathway, but also mediated by the Chk1, Mps1, Cga1 and Cgb1 pathways. This study provides insight into the roles of G-protein in mediating the anti-fungal fludioxonil response. A model is proposed for the interactions between the G-protein and MAPK signaling pathways.