Alternaria alternata, the casual agent of black rot of pear fruit, can sense and respond to the physicochemical cues from the host surface and form infection structures during infection. To evaluate the role of cyclic AMP-dependent protein kinase (cAMP-PKA) signaling in surface sensing of A. alternata, we isolated and functionally characterized the cyclic adenosine monophosphate-dependent protein kinase A catalytic subunit gene (AaPKAc). Gene expression results showed that AaPKAc was strongly expressed during the early stages of appressorium formation on hydrophobic surfaces. Knockout mutants ΔAaPKAc were generated by replacing the target genes via homologous recombination events. We found that intracellular cAMP content increased but PKA content decreased in ΔAaPKAc mutant strain. Appressorium formation and infection hyphae were reduced in the ΔAaPKAc mutant strain, and the ability of the ΔAaPKAc mutant strain to recognize and respond to high hydrophobicity surfaces and different surface waxes was lower than in the wild type (WT) strain. In comparison with the WT strain, the appressorium formation rate of the ΔAaPKAc mutant strain on high hydrophobicity and fruit wax extract surface was reduced by 31.6 and 49.3% 4 h after incubation, respectively. In addition, AaPKAc is required for the hypha growth, biomass, pathogenicity, and toxin production of A. alternata. However, AaPKAc negatively regulated conidia formation, melanin production, and osmotic stress resistance. Collectively, AaPKAc is required for pre-penetration, developmental, physiological, and pathological processes in A. alternata.
Calcium/calmodulin-dependent protein kinase (CaMK), a key downstream target protein in the Ca2+ signaling pathway of eukaryotes, plays an important regulatory role in the growth, development and pathogenicity of plant fungi. Three AaCaMKs (AaCaMK1, AaCaMK2 and AaCaMK3) with conserved PKC_like superfamily domains, ATP binding sites and ACT sites have been cloned from Alternaria alternata, However, their regulatory mechanism in A. alternata remains unclear. In this study, the function of the AaCaMKs in the development, infection structure differentiation and pathogenicity of A. alternata was elucidated through targeted gene disruption. The single disruption of AaCaMKs had no impact on the vegetative growth and spore morphology but significantly influenced hyphae growth, sporulation, biomass accumulation and melanin biosynthesis. Further expression analysis revealed that the AaCaMKs were up-regulated during the infection structure differentiation of A. alternata on hydrophobic and pear wax substrates. In vitro and in vivo analysis further revealed that the deletion of a single AaCaMKs gene significantly reduced the A. alternata conidial germination, appressorium formation and infection hyphae formation. In addition, pharmacological analysis confirmed that the CaMK specific inhibitor, KN93, inhibited conidial germination and appressorium formation in A. alternata. Meanwhile, the AaCaMKs genes deficiency significantly reduced the A. alternata pathogenicity. These results demonstrate that AaCaMKs regulate the development, infection structure differentiation and pathogenicity of A. alternata and provide potential targets for new effective fungicides.
To establish successful infections in host plants, pathogenic fungi must sense and respond to an array of stresses, such as oxidative stress. In this study, we systematically analyzed the effects of 30 mM H2O2 treatment on reactive oxygen species (ROS) metabolism in Alternaria alternata. Results showed that 30 mM H2O2 treatment lead to increased O2− generation rate and H2O2 content, and simultaneously, increased the activities and transcript levels of NADPH oxidase (NOX). The activities and gene expression levels of enzymes related with ascorbic acid-glutathione cycle (AsA-GSH cycle) and thioredoxin systems, including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), ascorbate peroxidase (AXP) and thioredoxin (TrxR), were remarkably enhanced by 30 mM H2O2 stress treatment. Additionally, 30 mM H2O2 treatment decreased the glutathione (GSH) content, whereas it increased the amount of oxidized glutathione (GSSG), dehydroascorbate (DHA) and ascorbic acid (AsA). These results revealed that cellular responses are required for oxidative stress tolerance of the necrotrophic fungus A. alternata.
Aims
Calmodulin (CaM), acts as a kind of multifunctional Ca2+ sensing protein, which is ubiquitous in fungi, is highly conserved across eukaryotes and is involved in the regulation of a range of physiological processes, including morphogenesis, reproduction and secondary metabolites biosynthesis. Our aim was to understand the characteristics and functions of AaCaM in Alternaria alternata, the causal agent of pear black spot.
Methods and results
A 450 bp cDNA sequence of AaCaM gene of A. alternata was cloned by the PCR homology method. Sequence analysis showed that this protein encoded by AaCaM was a stable hydrophilic protein and had a high similarity to Neurospora crassa (CAA50271.1) and other fungi. RT‐qPCR analysis determined that AaCaM was differentially upregulated during infection structural differentiation of A. alternata both on hydrophobic and pear wax extract‐coated surface, with a 3.37‐fold upregulation during the hydrophobic induced appressorium formation period (6 h) and a 1.46‐fold upregulation during the infection hyphae formation period (8 h) following pear wax induction. Pharmaceutical analysis showed that the CaM‐specific inhibitor, trifluoperazine (TFP), inhibited spore germination and appressorium formation, and affected toxins and melanin biosynthesis in A. alternata.
Conclusions
AaCaM plays an important role in regulating infection structure differentiation and secondary metabolism of A. alternata.
Significance and impact of study
Our study provides a theoretical basis for further in‐depth investigation of the specific role of AaCaM in the calcium signalling pathway underlying hydrophobic and pear wax‐induced infection structure differentiation and pathogenicity of A. alternata.
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