bZIP Transcription Factor UvATF21 Mediates Vegetative Growth,Conidiation,Stress Tolerance and Is Required for Full Virulence of Rice False Smut Fungus Ustilaginoidea virens

bZIP proteins are widely distributed in eukaryotic organisms and regulate a diverse range of physiological processes.Several bZIP proteins have previously been identified in Ustilaginoidea virens.However,the biological roles of these bZIP proteins in this pathogen are still unknown.Here,one of these bZIP protein coding genes,UvATF21,was functionally characterized.Targeted deletion of UvATF21resulted in reduced conidiation and pathogenicity despite of the increased vegetative growth.The deletion mutants also significantly decreased the sensitivity to osmotic and oxidative stresses.Interestingly,deletion of UvATF21 exhibited different performances to cell wall integrity stress.These results indicated that UvATF21 played crucial roles in vegetative growth,conidiation,stress response,and full virulence in U.virens.

Neurospora lca-1 Regulates Conidiation and Carotenoid Production

[Objective] This study aimed to investigate the regulatory mechanisms of carotenoid biosynthesis in Neurospora crassa. [Method] Gene knockout mutants pro- ducing less carotenoid were screened from 6 087 mutants; the yield of carotenoid and asexual spore was measured; finally fluorescent quantitative real-time PCR was adopted to analyze the transcription of genes related to carotenoid synthesis and asexual sporulation, [Result] Six knockout mutants produced less carotenoid. In one of them, the yield of both carotenoid and asexual spore reduced, because the gene which encodes an ATP-dependent chromatin remodelling complex ATPase chain ISW1 was knocked out. This gene was named /ca-1 in this study. And the /ca-1 deletion result- ed in a reduction of 88% in conidial production and a decrease of 81% in carotenoid production. [Conclusion] The Ica-1 positively regulates the carotenoid syn- thesis and asexual sporulation in N. crassa.

UvSMEK1,a Suppressor of MEK Null,Regulates Pathogenicity,Conidiation and Conidial Germination in Rice False Smut Fungus Ustilaginoidea virens

Rice false smut,which is caused by Ustilaginoidea virens,is an emerging disease of rice spikelets in rice-growing areas worldwide.However,the infection mechanism of U.virens on rice spikelets is still unclear.Here,we characterized a suppressor of mitogen-activated protein kinase kinase or ERK kinase(MEK)null(UvSMEKI)in U.virens that is conserved among filamentous fungi.Compared with wild type U.virens strain P-1,UvSMEKI deletion mutants were defective in pathogenicity and conidial germination.In addition,conidiation of UvSMEKI deletion mutants was significantly reduced on yeast extract tryptone(YT)plates,but inc「eased in YT broth compared with the wild type.Compared with UvSMEKI expression level during the vegetative mycelia and conidiation stages,UvSMEKI dramatically increased during infection of rice florets.Surprisingly,the UvSMEKI deletion mutants exhibited higher tolerance to H_(2)O_(2) and NaCl.In summary,presented evidence suggested that UvSMEKI positively regulated pathogenicity,conidial germination and conidiation in YT broth,and negatively regulated conidiation on YT medium and tolerance to oxidative and osmotic stresses.The results enhanee our understanding of the regulatory mechanism of pathogenicity of U.virens,and present a potential molecular target for blocking rice infection by U.virens.

Magnaporthe oryzae MTP1 gene encodes a type Ⅲ transmembrane protein involved in conidiation and conidial germination

In this study the MTP1 gene, encoding a type III integral transmembrane protein, was isolated from the rice blast fungus Magnaporthe oryzae. The Mtp 1 protein is 520 amino acids long and is comparable to the Ytp 1 protein of Saccharomyces cerevisiae with 46% sequence similarity. Prediction programs and MTP1-GFP （green fluorescent protein） fusion expression results indicate that Mtp 1 is a protein located at several membranes in the cytoplasm. The functions of the MTP1 gene in the growth and development of the fungus were studied using an MTP1 gene knockout mutant. The MTP1 gene was primarily expressed at the hyphal and conidial stages and is necessary for conidiation and conidial germination, but is not required for pathogenicity. The Amtpl mutant grew more efficiently than the wild type strain on non-fermentable carbon sources, implying that the MTP1 gene has a unique role in respiratory growth and carbon source use.

MoFLP1,encoding a novel fungal fasciclin-like protein,is involved in conidiation and pathogenicity in Magnaporthe oryzae

Fasciclin family proteins have been identified as cell adhesion molecules in various organisms. In this study, a novel Magnaporthe oryzae fasciclin-like protein encoding gene, named MoFLP1, was isolated from a subtractive suppressive cDNA library and functionally analyzed. Sequence analysis showed that the MoFLP1 gene contains an open reading frame (ORF) of 1050 nucleotides encoding 349 amino acids with a calculated molecular weight of 35.85 kDa and a pI of 7.76. The deduced MoFLP1 protein contains a 17-amino acid secretion signal sequence and an 18-amino acid sequence with the characteristics of a glycosylphosphotidylinositol (GPI) anchor additional signal at its N- and C-terminuses, respectively. Potential N-glycosylation sites and domains involving cell adhesion were also identified in MoFLP1. Sequence analysis and subcellular localization by the expression of MoFLP1-GFP fusion construct in M. oryzae indicated that the MoFLP1 protein is probably localized on the vacuole membrane. Two MoFLP1 null mutants generated by targeted gene disruption exhibited marked reduction of conidiation, conidial adhesion, appressorium turgor, and pathogenicity. Our results indicate that fasciclin proteins play important roles in fungal de-velopment and pathogenicity in M. oryzae.

The forced activation of asexual conidiation in Aspergillus niger simplifies bioproduction

Aspergillus niger is an efficient cell factory for organic acids production,particularly l-malic acid,through genetic manipulation.However,the traditional method of collecting A.niger spores for inoculation is labor-intensive and resource-consuming.In our study,we used the CRISPR-Cas9 system to replace the promoter of brlA,a key gene in Aspergillus conidiation,with a xylose-inducible promoter xylP in l-malic acid-producing A.niger strain RG0095,generating strain brlAxylP.When induced with xylose in submerged liquid culture,brlAxylP exhibited significant upregulation of conidiation-related genes.This induction allowed us to easily collect an abundance of brlAxylP spores(>7.1×106/mL)in liquid xylose medium.Significantly,the submerged conidiation approach preserves the substantial potential of A.niger as a foundational cellular platform for the biosynthesis of organic acids,including but not limited to l-malic acid.In summary,our study offers a simplified submerged conidiation strategy to streamline the preparation stage and reduce labor and material costs for industrial organic acid production using Aspergillus species.

Effects of Medium Nutrients on Fusarium oxysporum Schl. f.sp

[Objective] To study the effects of different culture conditions on the Fusarium oxysporurn SchL f. sp. [Method] Based on species identification of the pathogenic organism of Fusarium oxysporum Schl. f. sp, effects of different cultures and different nutrients on the mycelial growth and conidial production of Fusarium oxysporum SchL f. sp were studied. [Result] The mycelial growth and conidial pro- duction of Fusarium oxysporum SchL f. sp was different under different culture con- ditions. PDA medium was the most suitable medium for the mycelial growth and had the highest conidial production; and the mycelial grew the fastest on the medium with maltose as carbon source or peptone as nitrogen source, which also had the highest conidial production. [Conclusion] This study provided experimental basis for the study of Fusarium oxysporum SchL f. sp and also provided theoretical basis for the study and control of Fusarium oxysporum Schl. f. sp.

Vision and development in Trichoderma atroviride

Phototropism, the induction of carotenogenesis and reproductive structures, and resetting of the circadian rhythm are controlled by blue light. Trichoderma is used as a photomorphogenetic model due to its ability to conidiate upon exposure to light. In total darkness, T. atroviride grows indefinitely as a mycelium provided that nutrients are not limiting. However, nutrient deprivation and light trigger the conidiation process. A pulse of blue light given to a radially growing colony induces synchronous sporulation. A ring of conidiophores bearing green conidia is produced at what had been the colony perimeter at the time of the light pulse. All known responses to blue light in N. crassa are initiated by a couple of transcription factors encoded by the white-collar genes (wc -1 and wc-2). WC-1 and WC-2 bind to the promoters of light regulated genes to rapidly activate transcription in response to light. In T. atroviride the photolyase encoding gene phr1 undergoes fast transcriptional activation in response to light. The presence of putative WCC binding boxes in the promoter of phr1, suggested that light responses in Trichoderma could be under the control of white-collar homologues. We cloned two genes and demonstrated by gene replacement that both are essential for photoconidiation and photolyase gene expression. Therefore, they were named blue-light regulator one and two (blr1 and blr2). The BLR1 protein has all the characteristics of a blue-light photoreceptor. The generation of subtractive cDNA libraries allowed us to identify novel, BLR independent, light responses including the regulation of gene expression by blue-light. In addition, we recently initiated a Trichoderma ESTs sequencing project. Until now, we have sequenced above 3000 ESTs, from which we have obtained approximately 1800 unigenes. This unigene set was printed in microarrays and used to search for light induced genes. Twenty five clearly induced and around thirty repressed genes have been detected. Among this set we have found both blr dependent and independent blue light induced genes, strengthening our view of the existence of alternative light perception pathways. We also show the first evidence for the entry of Trichoderma into the conidiation process caused by mechanical injury, which remains unaltered in the mutants. Finally, an unprecedented crosstalk between light and glucose sensing was found involving the BLR1 and BLR2 proteins in the control of carbon deprivation induced conidiation.

Functional Characterization of a NEM1-Like Gene in Magnaporthe oryzae

Magnaporthe oryzae, a filamentous ascomycete fungus, is well known as the causal agent of rice blast. With the technology of suppression subtractive hybridization （SSH）, it was previously found that MGG_06001 （or named MoNEM1）, a gene of M. oryzae homologous to the NEM1 （nuclear envelope morphology protein 1） gene of baker＇s yeast （Saccharomyces cerevisiae）, is differentially expressed between the mature appressium and the conidium and mycelium. This study aimed to characterize the function of MoNEM1 gene by knocking it out using the method of target gene replacement. The AMoneml mutants exhibited reduced mycelial growth and conidiation. However, disruption of MoNEM1 gene does not affect the pathogenicity of M. oryzae on barley and rice.

Effects of MEK-Specific Inhibitor U0126 on the Conidial Germination,Appressorium Production,and Pathogenicity of Setosphaeria turcica

Systemic studies on the effects of mitogen-activated protein kinase （MAPK） signal transduction pathway on the growth and development of Setosphaeria turcica is helpful not only in understanding the molecular mechanism of pathogenhost interaction but also in the effective control of the diseases caused by S. turcica. U0126, the specific MEK inhibitor, is used to treat S. turcica before the observation of the conidial germination, appressorium production, and pathogenicity of the pathogen. There is no significant effect of U0126 on the colony morphology and mycelium growth of the pathogen. After treatment with U0126, the growth of mycelium and conidia are normal, but the conidial germination, appressorium production, and pathogenicity of S. turcica on susceptible corn leaves are significantly inhibited. Under the definite concentration scope, an increase in U0126 concentration increases the inhibition degree of conidial germination and appressorium production, but the inhibition degree decreases with elongation of treatment time. The conidial germination, appressorium production, and pathogenicity of S. turcica on susceptible corn leaves are regulated by the MAPK pathway inhibited by U0126.

Efficient Technique of Rapeseed and Morchella spp.Interplanting in Mountainous Areas

Aiming at the climatic characteristics of more rain and high humidity and cultivation habits of rapeseed in Tongren area,in terms of rapeseed and Morchella spp.interplanting,excellent varieties suitable for the local climate were selected,the formulas of culture substrate and nutrition bag were optimized to produce high-quality strains,soil pests were prevented and controlled,rapeseed was transplanted in time,Morchella spp.were sown in ditches,nutrition bags were placed simultaneously,thick soil was covered and fruiting ditches were left,and stress was arranged to promote growth of mycelia.The production cycle was 90-100 d.The yield of Morchella spp.reached 2250-3375 kg/ha,with a net profit of 6000-10000 yuan.

New records of Phaeoisaria triseptata and Spadicoides heterocolorata for Brazil

Phaeoisaria triseptata and Spadicoides heterocolorata are described,illustrated and discussed.Both species represent new records for Brazil.

Discovery and demonstration of the teleomorph of Beauve-ria bassiana (Bals.) Vuill.,an important entomogenous fungus

A Cordyceps specimen was collected in Anhui, China, a strain of Beauveria bassiana, an important ento-mopathogenic fungus for biological pest control, was isolated and their relationship was demonstrated by microcycle co-nidiation. The teleomorph is an undescribed species and is named Cordyceps bassiana.

VdPKS1 is required for melanin formation and virulence in a cotton wilt pathogen Verticillium dahliae

Verticillium dahliae is a soil-borne phytopathogenic fungus that causes vascular wilt disease in a broad range of hosts. This pathogen survives for many years in soil in the form ofmelanized microsclerotia. To investigate the melanin synthesis in V.. dahliae, we identified a polyketide synthase gene in V. dahliae, namely VdPKS1. PKS1 is known to involve in the dihydroxynaphthalene melanin synthesis pathway in many fungi. We found that VdPKS1 was required for melanin formation but not for microsclerotial production in E dahliae. The VdPKS1 gene-disruption mutant （vdpksl） formed melanin-deficient albino microsclerotia, which did not affect the fungal colonization in host tissues but significantly reduced the disease severity. Gene transcription analysis in the wild-type and the vdpks1 strains suggested that VdPKS1 gene-disruption influenced the expression of a series of genes involved in ethylene biosynthesis, microsclerotial formation and pathogenesis. Our results suggest that the VdPKS1-mediated melanin synthesis is important for virulence and developmental traits of E dahliae.

PKC-SWI6 signaling regulates asexual development,cell wall integrity,stress response,and lifestyle transition in the nematode-trapping fungus Arthrobotrys oligospora

Predatory fungi possess intricate signal transduction systems that regulate their development and support successful infection of the host.Herein,we characterized three components of the cell wall integrity-controlling pathway,namely protein kinase C(Ao PKC),SLT2-MAPK(Ao SLT2),and SWI6(Ao SWI6),in a representative nematode-trapping fungus Arthrobotrys oligospora,using gene disruption and multi-omics approaches.The phenotypic traits(such as mycelia development,conidiation,stress response,and trap morphogenesis) and metabolic profiles of ΔAopkc and ΔAoswi6 mutants were similar but differed from those of the ΔAoslt2 mutants.Transcriptomic analysis indicated that the genes differentially expressed in the absence of Aoswi6 were involved in DNA replication,repair,and recombination during trap formation.Moreover,the yeast two-hybrid assay showed that Ao PKC interacted with Ao SWI6,suggesting that in A.oligospora,PKC can directly regulate SWI6,bypassing the SLT2signaling cascade.Conclusively,our findings deepen our understanding of the regulatory mechanism of asexual development and lifestyle switching in nematode-trapping fungi.

Molecular data place the hyphomycetous lichenicolous genus Sclerococcum close to Dactylospora (Eurotiomycetes) and S. parmeliae in Cladophialophora (Chaetothyriales)

The lichenicolous anamorphic fungus Sclerococcum parmeliae was isolated in pure culture,and ITS,nuLSU and mtSSU sequences were obtained from these isolates.For comparison,sequences from S.sphaerale,the generic type,were obtained directly from freshly collected specimens.Phylogenetic analyses place S.sphaerale with species of Dactylospora and an unidentified lichen-inhabiting isolate in a strongly supported clade that is sister to a lineage comprising members of the Chaetothyriales and Pyrenulales.In contrast,S.parmeliae is inferred as a member of the Herpotrichiellaceae(Chaetothyriales)and belongs to a robustly supported clade that also includes species of Cladophialophora,Capronia semiimmersa,and Phialophora verrucosa.Within the Herpotrichiellaceae,S.parmeliae most closely resembles members of the anamorph genus Cladophialophora.Accordingly,we propose the transfer of S.parmeliae and the morphologically similar species S.cladoniae,S.hawksworthii and S.normandinae to Cladophialophora.A new lichenicolous species,Clad.megalosporae,collected twice on Megalospora in Florida and Papua New Guinea,is also described.

COP9 signalosome subunit PfCsnE regulates secondary metabolism and conidial formation in Pestalotiopsis fici

The COP9 signalosome(CSN) is a highly conserved multiprotein complex in all eukaryotes and involved in regulation of organism development. In filamentous fungi, several lines of evidence indicate that fungal development and secondary metabolism(SM) are mediated by the fifth subunit of CSN, called CsnE. Here we uncover a connection with CsnE and conidial formation as well as SM regulation in the plant endophytic fungus Pestalotiopsis fici. A homology search of the P. fici genome with CsnE, involved in sexual development and SM in Aspergillus nidulans, identified PfCsnE. Deletion of PfcsnE resulted in a mutant that stopped conidial production, but the conidia are recovered in a PfcsnE complemented strain. This indicates that PfCsnE is required for the formation of conidia. Secondary metabolite analysis demonstrated that the ΔPfcsnE strain produced more chloroisosulochrin, less ficiolide A production in comparison to wild type(WT). Transcriptome analysis of WT andΔPfcsnE strains indicated that PfcsnE impacts the expression levels of 8.37% of 14,797 annotated genes. Specifically, nine biosynthetic gene clusters(BGCs) were up-regulated and three BGCs were down-regulated by PfCsnE. Our results suggest that PfCsnE plays major roles in SM regulation and conidial development in P. fici.

Briancoppinsia,a new coelomycetous genus of Arthoniaceae(Arthoniales)for the lichenicolous Phoma cytospora,with a key to this and similar taxa

Morphological,anatomical,chemical and molecular data suggest that a relatively common lichenicolous coelomycete on Lecanora conizaeoides is conspecific with Phoma cytospora,previously known only from parmelioid lichens,and that further populations on Cladonia and Pertusaria belong to the same species.This species is distinguished from Phoma by several taxonomically important characters and obviously represents a previously unrecognized genus,for which the name Briancoppinsia is introduced.Phylogenetic analyses using nuLSU and mtSSU sequences of isolates obtained in pure culture suggest that the new genus belongs to the Arthoniaceae(Arthoniales).This is the first obligate lichenicolous,non-lichenized anamorph confirmed to belong to the Arthoniales based on molecular data.

Phylogenetic placement of lichenicolous Phoma species in the Phaeosphaeriaceae(Pleosporales,Dothideomycetes)

More than twenty species of lichenicolous fungi have been described in Phoma,a large anamorphic genus of primarily plant-associated pathogens with broad geographic distributions.We obtained nuclear and mitochondrial rDNA sequences from 19 fungal cultures isolated from specimens representing four described and two undescribed lichenicolous species in the genus.Our multilocus phylogeny indicates that lichenicolous Phoma species represent at least two phylogenetically distinct clades in the Phaeosphaeriaceae,one including a new species,Phoma puncteliae,isolated from a specimen of Punctelia rudecta collected inMaryland,USA,and another group of primarily lichenicolous species.This latter group includes four described lichenicolous Phoma species,an unidentified melanized rock fungus,and a new lichenicolous Phoma species isolated from Xanthomendoza species collected in Canada that we are naming P.xanthomendozae.Some specimens in this clade collected from different lichen genera and species were found to be very similar genetically,which calls into question the recent practice of recognizing lichenicolous Phoma species mainly by differences in host preference.