Cyclic AMP-dependent protein kinase A negatively regulates conidia formation by the tangerine pathotype of Alternaria alternata

The necrotrophic fungal pathogen Alternaria alternata causes brown spot diseases in many citrus cultivars. The FUS3 and SLT2 mitogen-activated protein kinases (MAPK)-mediated signaling pathways have been shown to be required for conidiation. Exogenous application of cAMP to this fungal pathogen decreased conidia formation considerably. This study determined whether a cAMP-activated protein kinase A (PKA) is required for conidiation. Using loss-of-function mutations in PKA catalytic and regulatory subunit-coding genes, we demonstrated that PKA negatively regulates conidiation. Fungal mutants lacking PKA catalytic subunit gene (PKA ^cat ) reduced growth, lacked detectable PKA activity, and produced higher amounts of conidia compared to wild-type. Introduction of a functional copy of PKA ^cat into a null mutant partially restored PKA activity and produced wild-type level of conidia. In contrast, fungi lacking PKA regulatory subunit gene (PKA ^reg ) produced detectable PKA activity, exhibited severe growth reduction, formed swelling hyphal segments, and produced no mature conidia. Introduction of the PKA ^reg gene to a regulatory subunit mutant restored all phenotypes to wild type. PKA ^reg -null mutants induced fewer necrotic lesions on citrus compared to wild-type, whereas PKA ^cat mutant displayed wild-type virulence. Overall, our studies indicate that PKA and FUS3-mediated signaling pathways apparently have very different roles in the regulation of conidia production and A. alternata pathogenesis in citrus.     

How the Pathogenic Fungus Alternaria alternata Copes with Stress via the Response Regulators SSK1 and SHO1

The tangerine pathotype of Alternaria alternata is a necrotrophic fungal pathogen causing brown spot disease on a number of citrus cultivars. To better understand the dynamics of signal regulation leading to oxidative and osmotic stress response and fungal infection on citrus, phenotypic characterization of the yeast SSK1 response regulator homolog was performed. It was determined that SSK1 responds to diverse environmental stimuli and plays a critical role in fungal pathogenesis. Experiments to determine the phenotypes resulting from the loss of SSK1 reveal that the SSK1 gene product may be fulfilling similar regulatory roles in signaling pathways involving a HOG1 MAP kinase during ROS resistance, osmotic resistance, fungicide sensitivity and fungal virulence. The SSK1 mutants display elevated sensitivity to oxidants, fail to detoxify H₂O₂ effectively, induce minor necrosis on susceptible citrus leaves, and displays resistance to dicarboximide and phenylpyrrole fungicides. Unlike the SKN7 response regulator, SSK1 and HOG1 confer resistance to salt-induced osmotic stress via an unknown kinase sensor rather than the “two component” histidine kinase HSK1. SSK1 and HOG1 play a moderate role in sugar-induced osmotic stress. We also show that SSK1 mutants are impaired in their ability to produce germ tubes from conidia, indicating a role for the gene product in cell differentiation. SSK1 also is involved in multi-drug resistance. However, deletion of the yeast SHO1 (synthetic high osmolarity) homolog resulted in no noticeable phenotypes. Nonetheless, our results show that A. alternata can sense and react to different types of stress via SSK1, HOG1 and SKN7 in a cooperative manner leading to proper physiological and pathological functions.     

Identification and functional analysis of Penicillium digitatum genes putatively involved in virulence towards citrus fruit

The fungus Penicillium digitatum, the causal agent of green mould rot, is the most destructive post‐harvest pathogen of citrus fruit in Mediterranean regions. In order to identify P. digitatum genes up‐regulated during the infection of oranges that may constitute putative virulence factors, we followed a polymerase chain reaction (PCR)‐based suppression subtractive hybridization and cDNA macroarray hybridization approach. The origin of expressed sequence tags (ESTs) was determined by comparison against the available genome sequences of both organisms. Genes coding for fungal proteases and plant cell wall‐degrading enzymes represent the largest categories in the subtracted cDNA library. Northern blot analysis of a selection of P. digitatum genes, including those coding for proteases, cell wall‐related enzymes, redox homoeostasis and detoxification processes, confirmed their up‐regulation at varying time points during the infection process. Agrobacterium tumefaciens‐mediated transformation was used to generate knockout mutants for two genes encoding a pectin lyase (Pnl1) and a naphthalene dioxygenase (Ndo1). Two independent P. digitatum Δndo1 mutants were as virulent as the wild‐type. However, the two Δpnl1 mutants analysed were less virulent than the parental strain or an ectopic transformant. Together, these results provide a significant advance in our understanding of the putative determinants of the virulence mechanisms of P. digitatum.     

Selection of mutants resistant to Alternaria blotch from in vitro -cultured apple shoots irradiated with X- and γ-rays

We produced mutants resistant to Alternaria blotch disease in several cultivars of apple (Malus × domestica Borkh.) by irradiation with X- or γ-rays. An efficient in vitro assay method was established using chemically-synthesized AM-toxin I of Alternaria alternata (Fr.) Keissler to screen for mutants resistant to Alternaria blotch disease. The frequency of necrotic lesions was investigated by applying various concentrations of AM-toxin I to leaf discs of the first, third, and fifth leaves from the shoot apex of several apple cultivars, including Jonathan, Fuji, Oorin, and Indo. In vitro-grown apple shoots of susceptible cultivars were then treated with various doses of X- or γ-ray irradiation. Several mutants resistant to AM-toxin I were obtained by combining the techniques for tissue culture of apple shoots with the AM-toxin I screening method. Following a repeat second screening test with AM-toxin I, mutant plants were sprayed with a spore suspension of A. alternata and found resistant to be the fungal pathogen. These mutants showed normal phenotypic appearance, and so far, no difference has been observed between the original plants and mutants except for the susceptibility to Alternaria blotch.     

Host Physiology and Pathogenic Variation of Cochliobolus heterostrophus Strains with Mutations in the G Protein Alpha Subunit, CGA1

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.