GPH1 is involved in glycerol accumulation in the three-dimensional networks of the nematode-trapping fungus <Emphasis Type="Italic">Arthrobotrys oligospora</Emphasis>

Turgor is very important for the invasive growth of fungal pathogens. Glycerol, a highly osmotic solvent, is considered to play an important role in turgor generation. The nematophagous fungus Arthrobotrys oligospora mainly lives as a saprophyte. In the presence of nematodes, A. oligospora enters the parasitic stage by forming three-dimensional networks (traps) to capture nematodes. In A. oligospora, we found that glycerol accumulated during nematode-induced trap formation. We demonstrated that deleting gph1, which encodes glycogen phosphorylase, decreased the glycerol content, compared with that of a wild-type strain. Although the number of traps induced by nematodes was not affected in the Δgph1 mutant, the capture rate was lower. Meanwhile, deleting gph1 also affected the growth rate and conidiation capacity of the fungus. These results indicate that glycerol derived from GPH1 is essential for the full virulence of A. oligospora against nematodes.     

Ion Beam Mutagenesis in Arthrobotrys oligospora Enhances Nematode-Trapping Ability

The nematode-trapping fungus Arthrobotrys oligospora is able to produce extracellular protease that degrades the body walls of parasitic nematode larvae found in livestock and immobilizes the nematodes. Our aim was to obtain a strain of A. oligospora with a strong ability to trap nematodes by production of high levels of extracellular protease. A wild type strain of A. oligospora was subjected to mutagenic treatments involving low-energy ion beam implantation to generate mutants. Among these mutants, A. oligospora N showed high efficiency in trapping nematodes and was also able to secrete more extracellular protease, helping it to penetrate and digest the body walls of larvae. This work represents the first application of low-energy ion beams to generate mutations in a nematode-trapping fungus, and provides a new method of obtaining a fungus with high potential application.     

Coding the α-subunit of SNF1 kinase,Snf1 is required for the conidiogenesis and pathogenicity of the Alternaria alternata tangerine pathotype

To well cope with various external carbon sources, fungi have evolved an adaptive mechanism to overcome the adversity of carbon source deficiency. The sucrose non-fermenting (SNF1) protein kinase mainly mediates the utilization of non-fermentable carbon sources. In this study, we determined the function of Snf1, coding the α-subunit of SNF1 kinase, in the phytopathogenic fungus Alternaria alternata via analyzing the Snf1 deletion mutants (ΔAasnf1). Aasnf1 is required for growth, development of aerial mycelium, and conidiation. Results of pathogenicity test showed that ΔAasnf1 induced smaller lesions on detached citrus leaves. Moreover, in the carbon utilization assay, ΔAasnf1 showed growth inhibition on the minimal medium supplemented with polygalacturonic acid, sucrose or alcohol as the only carbon source. Compared to the wild type, ΔAasnf1 also exhibited stronger resistance to cell wall stressors of sodium dodecyl sulfate and congo red. In conclusion, Aasnf1 played important roles in the carbon utilization, vegetative growth, conidiation, cell wall functions and pathogenicity of A. alternata. This study is the first report on the functions of Aasnf1 and our results suggest that Snf1 is critical for the conidiogenesis and pathogenesis of the A. alternata tangerine pathotype.     

The NADPH oxidase AoNoxA in <Emphasis Type="Italic">Arthrobotrys oligospora</Emphasis> functions as an initial factor in the infection of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Reactive oxygen species (ROS) produced by NADPH oxidases can serve as signaling molecules to regulate a variety of physiological processes in multi-cellular organisms. In the nematophagous fungus Arthrobotrys oligospora, we found that ROS were produced during conidial germination, hyphal extension, and trap formation in the presence of nematodes. Generation of an AoNoxA knockout strain demonstrated the crucial role of NADPH oxidase in the production of ROS in A. oligospora, with trap formation impaired in the AoNoxA mutant, even in the presence of the nematode host. In addition, the expression of virulence factor serine protease P186 was up-regulated in the wild-type strain, but not in the mutant strain, in the presence of Caenorhabditis elegans. These results indicate that ROS derived from AoNoxA are essential for full virulence of A. oligospora in nematodes.     

An endogenous rhythm of trap formation in the nematophagous fungus Arthrobotrys oligospora

In the predacious fungus Arthrobotrys oligospora Fres., the number and distribution of traps formed after the addition of living nematodes to the colonies were determined. At 21°C the traps were formed periodically; the mean period was 42.3±0.8 h. The periodicity was independent of light-dark (LD) cycles of 24 h (10:14). Temperature influenced the hyphal elongation but did not affect the periodic trap formation; at lower temperatures the peaks of trap formation were close together, showing partial overlapping. Induction of rhythmic mycelial growth and conidiation by chemical means was effective only in LD-cycles. The latter diurnal rhythm was weakly correlated with the trap formation and did not affect the endogenous period of approximately 42 h.Abbreviations LD light-dark - DD continuous darkness - LNM low-nutrient medium - CMA corn meal agar     

Evaluation of biocontrol potential of Arthrobotrys oligospora against Meloidogyne graminicola and Rhizoctonia solani in Rice (Oryza sativa L.)

The nematode trapping and mycoparasitic potential of Arthrobotrys oligospora was tested in vitro against Meloidogyne graminicola and Rhizoctonia solani, respectively. Five isolates of A. oligospora were isolated from different locations of India. Diversity of the trapping structures is large and highly dependent on the environmental condition and nature of the fungus. In A. oligospora, a three-dimensional adhesive net (in response to nematode) and hyphal coils developed around the hyphae of R. solani. In vitro trap formation and predacity were tested against second-stage juveniles of M. graminicola (J₂) and the interactions between A. oligospora and R. solani were recorded. Under field conditions, we demonstrated the biocontrol potential of A. oligospora against R. solani causing sheath blight of rice (Oryza sativa) for the first time. All the isolates of A. oligospora parasitized and killed M. graminicola and R. solani. Application of A. oligospora, isolate VNS-1, in soil infested with M. graminicola and R. solani reduced the number of root knot by 57.58–62.02%, sheath blight incidence by 55.68–59.32% and lesion length by 54.91–66.66% under green house and miniplot (field) conditions. Applications of A. oligospora to the soil increased plant growth: shoot length by 56.4–68.8%, root length by 44.0–54.55%, fresh weight of shoot and root by 62.91–65.4% and 38.9–44.19%, respectively, as compared to the plants grown in nematode infested soil.     

An adaptor protein BmSte50 interacts with BmSte11 MAPKKK and is involved in host infection,conidiation, melanization,and sexual development in Bipolaris maydis

A mitogen-activated protein kinase (MAPK) signaling pathway regulates specialized cellular responses to external stimuli. In Bipolaris maydis, a Chk1 MAPK orthologous to Fus3/Kss1 MAPKs of Saccharomyces cerevisiae is known to regulate various developmental processes, including the formation of appressoria. However, upstream factors that regulate the Chk1 cascade have not been well clarified. In this study, we identified and characterized the BmSte50 gene, an ortholog of the yeast Ste50 in B. maydis. Our yeast two-hybrid assay indicated that BmSte50 interacts with a MAPK kinase kinase BmSte11, a component of the Chk1 cascade. ΔBmSte50 strains exhibited a loss of pathogenicity due to a lack of appressorial formation. The mutants also showed a reduction in melanization, conidial production, and aerial-mycelial and sexual development. Such phenotypes of the mutants were consistent with those of the Chk1 cascade gene mutants previously reported. In addition, ΔBmSte50 strains indicated lower conidial germination efficiency than the wild type. Notably, a significant number of ΔBmSte50 conidia could be germinated, while the Chk1 cascade gene mutants were reported to lack conidial germination ability. Our results suggested that BmSte50 may act as an adaptor protein for the Chk1 cascade and is involved in the regulation of various cellular processes.     

Glutamate synthase MoGlt1‐mediated glutamate homeostasis is important for autophagy,virulence and conidiation in the rice blast fungus

Glutamate homeostasis plays a vital role in central nitrogen metabolism and coordinates several key metabolic functions. However, its function in fungal pathogenesis and development has not been investigated in detail. In this study, we identified and characterized a glutamate synthase gene MoGLT1 in the rice blast fungus Magnaporthe oryzae that was important to glutamate homeostasis. MoGLT1 was constitutively expressed, but showed the highest expression level in appressoria. Deletion of MoGLT1 resulted in a significant reduction in conidiation and virulence. The ΔMoglt1 mutants were defective in appressorial penetration and the differentiation and spread of invasive hyphae in penetrated plant cells. The addition of exogenous glutamic acid partially rescued the defects of the ΔMoglt1 mutants in conidiation and plant infection. Assays for MoAtg8 expression and localization showed that the ΔMoglt1 mutants were defective in autophagy. The ΔMoglt1 mutants were delayed in the mobilization of glycogens and lipid bodies from conidia to developing appressoria. Taken together, our results show that glutamate synthase MoGlt1‐mediated glutamate homeostasis is important for pathogenesis and development in the rice blast fungus, possibly via the regulation of autophagy.     

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.     

Interaction of nematodes with nematophagus fungi: induction of trap formation, attraction and detection of attractants

Induction of trap formation in Arthrobotrys oligospora, A. conoides and Monacrosporium cystosporum was studied during a 24 h period in the presence of the free-living nematodes Panagrellus redivivus on various nutrient media. A definite pattern of trap formation was observed in these fungi. Low nutrient mineral salt medium had the most pronounced effect on trap formation. Attraction and repulsion of P. redivivus towards these three nematophagous and three non-nematophagous fungi was studied. The nematodes were attracted towards three nematophagous and one non-nematophagous fungi, the other two repelled them. Attractants of the nematophagous fungi were determined by thin-layer chromatography. In the case of A. oligospora and M. cystosporum, four spots were traced, whereas in A. conoides, five spots were detected.     

PdSNF1, a sucrose non-fermenting protein kinase gene, is required for Penicillium digitatum conidiation and virulence

The sucrose non-fermenting protein kinase 1 gene (SNF1) regulates the derepression of glucose-repressible genes in microorganisms. In this study, we cloned an ortholog of SNF1 from Penicillium digitatum and characterized its functions through a gene knock-out strategy. Growth of the PdSNF1 mutant (ΔPdSNF1) on the synthetic medium (SM) supplemented with pectin or polygalacturonic acid was severely disturbed. The appearance of disease symptoms on the ΔPdSNF1 mutant-inoculated citrus fruits was significantly delayed as well. The expression levels of the cell wall-degrading enzyme (CWDE) genes (e.g., XY1, PL1, PNL1, and EXPG2) after pectin induction were up-regulated in wild type, but unchanged or less up-regulated in the ΔPdSNF1 mutant. During infection in citrus fruit, the up-regulation of XY1 was delayed in the ΔPdSNF1 mutant. Disruption of PdSNF1 also resulted in impaired conidiation and caused malformation of the conidiophore structures. In addition, the expression of BrlA, a gene that regulates conidiophore development, was significantly impaired in the ΔPdSNF1 mutant. However, the expression of FadA, encoding the α-subunit of a heterotrimeric G protein, was up-regulated in this mutant. Collectively, our results demonstrate that the PdSNF1 plays a role in adapting P. digitatum to alternative carbon sources. Its involvements in the virulence of P. digitatum is probably via regulation of the expression of CWDE genes; and it is also involved in conidiation, probably through activation of the conidiation signaling pathway while inactivating the mycelial growth-signaling pathway.     

Calcineurin modulates growth,stress tolerance,and virulence in Metarhizium acridum and its regulatory network

Calcineurin is highly conserved and regulates growth, conidiation, stress response, and pathogenicity in fungi. However, the functions of calcineurin and its regulatory network in entomopathogenic fungi are not clear. In this study, calcineurin was functionally analyzed by deleting the catalytic subunit MaCnA from the entomopathogenic fungus Metarhizium acridum. The ΔMaCnA mutant had aberrant, compact colonies and blunt, shortened hyphae. Conidia production was reduced, and phialide differentiation into conidiogenous cells was impaired in the ΔMaCnA mutant. ΔMaCnA had thinner cell walls and greatly reduced chitin and β-1,3-glucan content compared to the wild type. The ΔMaCnA mutant was more tolerant to cell wall-perturbing agents and elevated or decreased exogenous calcium but less tolerant to heat, ultraviolet irradiation, and caspofungin than the wild type. Bioassays showed that ΔMaCnA had decreased virulence. Digital gene expression profiling revealed that genes involved in cell wall construction, conidiation, stress tolerance, cell cycle control, and calcium transport were downregulated in ΔMaCnA. Calcineurin affected some components of small G proteins, mitogen-activated protein kinase, and cyclic AMP (cAMP)-protein kinase A signaling pathways in M. acridum. In conclusion, our results gave a global survey of the genes downstream of calcineurin in M. acridum, providing molecular explanations for the changes in phenotypes observed when calcineurin was deleted.