SreC-dependent adaption to host iron environments regulates the transition of trophic stages and developmental processes of Curvularia lunata.

Plant pathogens are challenged by host-derived iron starvation or excess during infection, but the mechanism of plant pathogens rapidly adapting to the dynamic host iron environments to assimilate iron for invasion and colonization remains largely unexplored. Here, we found that the GATA transcription factor SreC in Curvularia lunata is required for virulence and adaption to the host iron excess environment. SreC directly binds to the ATGWGATAW element in an iron-dependent manner to regulate the switch between different iron assimilation pathways, conferring adaption to host iron environments in different trophic stages of C. lunata. SreC also regulates the transition of trophic stages and developmental processes in C. lunata. SreC-dependent adaption to host iron environments is essential to the infectious growth and survival of C. lunata. We also demonstrate that CgSreA (a SreC orthologue) plays a similar role in Colletotrichum graminicola. We conclude that Sre mediates adaption to the host iron environment during infection, and the function is conserved in hemibiotrophic fungi.

An efficient targeted gene deletion approach for Cochliobolus heterostrophus using Agrobacterium tumefaciens-mediated transformation.

Cochliobolus heterostrophus is a plant pathogenic fungus of southern corn leaf blight, which has been regarded as a model necrotrophic plant pathogen. Many methods have been developed to knock out targeted genes in C. heterostrophus, of which the most widely-used one is protoplast-mediated transformation. However, there are several problems of this method associated with protoplast preparation, DNA product, time consumption, or high cost. In this study, a highly efficient target gene deletion approach in C. heterostrophus was established and optimized, based on Agrobacterium tumefaciens-mediated transformation (ATMT); the transformation efficiency of this approach was 85-88 transformants per 105 conidia, and the homologous recombination efficiency was approximately 68.3%. Furthermore, six gene knockout mutants of C. heterostrophus were obtained using this ATMT method. The phenotypes of this fungus altered in the mutant strains, and the virulence of the mutants significantly reduced compared to of the wild type strain. Taken together, this ATMT system established in this study can be used as a genetic manipulation tool for C. heterostrophus, to better understand the functions of genes and its relation to virulence.

Highly efficient gene knockout system in the maize pathogen Colletotrichum graminicola using Agrobacterium tumefaciens-mediated transformation (ATMT).

Colletotrichum graminicola, a hemibiotrophic pathogenic fungus, is the causal agent of anthracnose of maize, which causes significant yield losses worldwide, especially in warm and humid maize production regions. An efficient targeted genes knockout protocol is crucial to explore molecular mechanisms of fungal virulence to the host. In this study, we established a gene knockout transformation system by employing Agrobacterium tumefaciens-mediated transformation to knockout genes in M 1.001 strain of C. graminicola. The conidia germination status, induction medium type, and ratio of Agrobacterium cell and conidia suspension were optimized for the knockout of CgBRN1(OR352905), a gene relating to the fungal melanin biosynthesis pathway. Additionally, CgPKS18 (OR352906) and CgCDC25 (OR352903) were knocked out to test the applicability of the gene knockout transformation system. In this established system, transformation efficiency was 176 transformants per 1 × 105 conidia and the homologous recombination efficiency was 53.3 to 75%. Furthermore, disease index, lesion number and lesion size caused by the three above-mentioned mutant strains were found to be reduced significantly compared to the wild-type strain, which indicated reduction in fungal virulence due to the lack of those genes.

Iron redistribution induces oxidative burst and resistance in maize against Curvularia lunata.

MAIN CONCLUSION: ΔClnps6 induced iron redistribution in maize B73 leaf cells and resulted in reactive oxygen species (ROS) burst to enhance plant resistance against Curvularia lunata. Iron is an indispensable co-factor of various crucial enzymes that are involved in cellular metabolic processes and energy metabolism in eukaryotes. For this reason, plants and pathogens compete for iron to maintain their iron homeostasis, respectively. In our previous study, ΔClnps6, the extracellular siderophore biosynthesis deletion mutant of Curvularia lunata, was sensitive to exogenous hydrogen peroxide and virulence reduction. However, the mechanism was not studied. Here, we report that maize B73 displayed highly resistance to ΔClnps6. The plants recruited more iron at cell wall appositions (CWAs) to cause ROS bursts. Intracellular iron deficiency induced by iron redistribution originated form up-regulated expression of genes involved in intracellular iron consumption in leaves and absorption in roots. The RNA-sequencing data also showed that the expression of respiratory burst oxidase homologue (ZmRBOH4) and NADP-dependent malic enzyme 4 (ZmNADP-ME4) involved in ROS production was up-regulated in maize B73 after ΔClnps6 infection. Simultaneously, jasmonic acid (JA) biosynthesis genes lipoxygenase (ZmLOX), allene oxide synthase (ZmAOS), GA degradation gene gibberellin 2-beta-dioxygenase (ZmGA2OX6) and ABA degradation genes abscisic acid hydroxylase (ZmABH1, ZmABH2) involved in iron homeostasis were up-regulated expression. Ferritin1 (ZmFER1) positive regulated maize resistance against C. lunata via ROS burst under Fe-limiting conditions. Overall, our results showed that iron played vital roles in activating maize resistance in B73-C. lunata interaction.

ClNOX1/ClNOXR-mediated MAPK and cAMP-PKA signalling pathways and ROS metabolism are involved in Curvularia lunata sexual reproduction and host infection.

NADPH oxidases (NOXs) and hydrogen peroxide (H2 O2 ) are involved in physiological and pathological processes, and cell fate decisions in organisms. However, regulatory mechanism of NOXs and the role of H2 O2 on fungal sexual reproduction and host infection remain largely unexplored. Here, we identified ROS metabolic genes and key signalling genes of MAPK and cAMP-PKA pathways in Curvularia lunata, which were NOX ClNOX1 and ClNOXR, superoxide dismutase ClSOD1 and catalase ClCAT4, redox-regulated transcription factor ClAP1, Ras small GTPases Clg2P, pheromone-response MAPK ClK1 and cAMP-PKA ClSCHA, and characterized the functions of these genes. The results showed that ClNOX1 localized to the plasma membrane. ClNOX1 and ClNOXR were involved in sexual reproduction and host infection via ClNOX1/ClNOXR-derived H2 O2 as well as MAPK and cAMP-PKA signalling pathways. H2 O2 acted as a signalling molecule to regulate sexual reproduction and host infection in C. lunata.

The key iron assimilation genes ClFTR1, ClNPS6 were crucial for virulence of Curvularia lunata via initiating its appressorium formation and virulence factors.

Iron is virtually an essential nutrient for all organisms, to understand how iron contributes to virulence of plant pathogenic fungi, we identified ClFTR1 and ClNPS6 in maize pathogen Curvularia lunata (Cochliobolus lunatus) in this study. Disruption of ClNPS6 significantly impaired siderophore biosynthesis. ClFTR1 and ClNPS6 did mediate oxidative stress but had no significant impact on vegetative growth, conidiation, cell wall integrity and sexual reproduction. Conidial germination delayed and appressoria formation reduced in ΔClftr1 comparing with wild type (WT) CX-3. Genes responsible for conidial germination, appressoria formation, non-host selective toxin biosynthesis and cell wall degrading enzymes were also downregulated in the transcriptome of ΔClftr1 and ΔClnps6 compared with WT. The conidial development, toxin biosynthesis and polygalacturonase activity were impaired in the mutant strains with ClFTR1 and ClNPS6 deletion during their infection to maize. ClFTR1 and ClNPS6 were upregulated expression at 12-24 and 48-120 hpi in WT respectively. ClFTR1 positively regulated conidial germination, appressoria formation in the biotrophy-specific phase. ClNPS6 positively regulates non-host selective toxin biosynthesis and cell wall degrading enzyme activity in the necrotrophy-specific phase. Our results indicated that ClFTR1 and ClNPS6 were key genes of pathogen known to conidia development and virulence factors.

Virulence and Molecular Diversity in the Kabatiella zeae Population Causing Maize Eyespot in China.

Maize eyespot, caused by Kabatiella zeae, has become a major yield-limiting factor in maize planting areas in northeast China. Limited information is available on pathotypes, virulence, and the genetic diversity of the K. zeae population. We analyzed virulence and genetic diversity of 103 K. zeae isolates collected from six provinces in China with differential hosts and the amplified fragment length polymorphism (AFLP) technique, respectively. To evaluate the virulence, 103 isolates were inoculated on nine differential hosts (maize inbred lines)-E28, Shen137, Qi319, B73, Danhuang34, Zi330, Mo17, Huangzaosi, and CN165-and grouped into 23 pathotypes and three virulence groups according to the coded triplet nomenclature system on differential hosts. AFLP analysis resolved the set of isolates into four genetic diversity clusters (DICE similarity values of 76%). Genetic variation of K. zeae among and between pathotypes revealed that the pathogen population had a high genotypic diversity. The correlation between pathotypes, virulence, and genetic diversity grouping was low. A correlation between AFLP groups and geographic locations was detected.

NADPH Oxidase ClNOX2 Regulates Melanin-Mediated Development and Virulence in Curvularia lunata.

The role of NADPH oxidases (NOXs) in pathogenesis and development in the Curvularia leaf spot agent Curvularia lunata remains poorly understood. In this study, we identified C. lunata ClNOX2, which localized to the plasma membrane and was responsible for reactive oxygen species (ROS) generation. Scavenging the ROS production inhibited the conidial germination and appressorial formation. The ClNOX2 and ClBRN1 deletion mutants were defective in 1,8-dihydroxynaphthalene (DHN) melanin accumulation, appressorial formation, and cellulase synthesis and exhibited lower virulence. However, disruption of the ClNOX2 and ClBRN1 genes facilitated hyphal growth, enhanced stress adaptation to cell-wall-disrupting agents, and promoted developmental processes such as conidiation, conidial germination, and pseudothecium and ascus formation. Interestingly, loss of ClM1, the cell wall integrity (CWI) mitogen-activated protein kinase gene in C. lunata, led to morphology and pathogenicity phenotypes similar to ClNOX2 and ClBRN1 deletion mutants such as abnormal conidia, fewer appressoria, less melanin, increased hyphal growth, and enhanced tolerance to Congo red (CR). These results indicated that the ClNOX2 gene plays an important role in C. lunata development and virulence via regulating intracellular DHN melanin biosynthesis. Quantitative reverse-transcription PCR revealed that the ClNOX2-related ROS signaling pathway and ClM1-mediated CWI signaling pathway are cross-linked in regulating DHN melanin biosynthesis. Our findings provide new insights into how ClNOX2 participates in pathogenesis and development in hemibiotrophic plant fungal pathogens.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Virulence, Molecular Diversity, and Mating Type of Curvularia lunata in China.

Curvularia leaf spot (CuLS), caused by Curvularia lunata, is a devasting foliar disease in the maize-growing regions of China. Resistant varieties were widely planted in these regions in response to CuLS. However, over time, C. lunata has gradually adapted to the selective pressure and, in recent years, the incidence of CuLS has increased. To assess the correlation between virulence and genetic diversity, a total of 111 isolates was collected from 15 maize-growing regions located in nine provinces in China. These isolates were evaluated for virulence on maize using nine differential hosts: Shen135, CN165, Mo17, Luyuan92, 78599, Ye478, B73, E28, and Huangzaosi. To evaluate the genetic diversity, 657 polymorphic amplified fragment length polymorphism markers were generated. Results showed that the isolates could be grouped into three pathotypes according to the phenotypic expression of the differential inbred lines. Isolates were clustered into two genetic diversity groups and further divided into subgroups. However, the correlation between virulence and genetic diversity grouping was low. Also, there was a low correlation observed between pathotype and geographic distribution. The ratio of mating type I to mating type II for all isolates was close to 3:4.

Agrobacterium tumefaciens-mediated transformation as an efficient tool for insertional mutagenesis of Kabatiella zeae.

Agrobacterium tumefaciens-mediated transformation (ATMT) has been widely used in filamentous fungi. In this study, an efficient Agrobacterium tumefaciens-mediated transformation approach was developed for the plant pathogenic fungus, Kabatiella zeae, the causative pathogen of eyespot in maize. Five parameters were selected to optimize efficiencies of transformation. A. tumefaciens concentration, conidia concentration of K. zeae and mixing ratio of A. tumefaciens and K. zeae were found to exert a significant influence on all parameters. Transformants emitted green fluorescence under fluorescence microscopy. The presence of mitotically stable hygromycin resistance gene (hph) integration in the genome was confirmed by PCR. Up to 148 transformants per 107 conidia could be obtained under optimal conditions. In this way, ATMT approach is an efficient tool for insertional mutagenesis of K. zeae.

Using overlap-extension PCR technique to fusing genes for constructing recombinant plasmids.

Overlap-extension PCR is a method for splice of gene segments to produce focused fragments for constructing recombinant plasmid, but its complexity limits its application. To simplify the protocol and to improve the effectiveness, we employed gradient temperatures to replace the single annealing temperature in the thermo-cycling program, and optimize the templates ratio. The concentration of each fragment was adjusted to 10 ng µl-1 . Fragment concentration ratio was the inverse of the fragment size ratio. The products of fused segments were 2000-5000 bp in length using the revised one-step method. This method splices effective two or more fragments to fused gene and produce recombinant plasmid.

Agrobacterium tumefaciens-mediated transformation as an efficient tool for insertional mutagenesis of Cercospora zeae-maydis.

An efficient Agrobacterium tumefaciens-mediated transformation (ATMT) approach was developed for the plant pathogenic fungus, Cercospora zeae-maydis, which is the causative agent of gray leaf spot in maize. The transformation was evaluated with five parameters to test the efficiencies of transformation. Results showed that spore germination time, co-cultivation temperature and time were the significant influencing factors in all parameters. Randomly selected transformants were confirmed and the transformants were found to be mitotically stable, with single-copy T-DNA integration in the genome. T-DNA flanking sequences were cloned by thermal asymmetric interlaced PCR. Thus, the ATMT approach is an efficient tool for insertional mutagenesis of C. zeae-maydis.

Efficient gene knockout in the maize pathogen Setosphaeria turcica using Agrobacterium tumefaciens-mediated transformation.

Setosphaeria turcica, a hemibiotrophic pathogenic dothideomycete, is the causal agent of Northern Leaf Blight of maize, which periodically causes significant yield losses worldwide. To explore molecular mechanisms of fungal pathogenicity and virulence to the host, an efficient targeted gene knockout transformation system using Agrobacterium tumefaciens was established with field collected strains. The starting materials, incubation time, induction medium type, Agrobacterium cell density, and method of co-incubation were optimized for deletion of 1,3,8-trihydroxynaphthalene reductase, a gene in the melanin biosynthesis pathway, as a test case. Four additional genes were deleted in two different S. turcica field isolates to confirm robustness of the method. One of these mutant strains was reduced in virulence compared with the wild-type strain when inoculated on susceptible maize. Transformation efficiency was ≈20 ± 3 transformants per 1× 10(6) germlings and homologous recombination efficiency was 33.3 to 100%.

Comparative genome structure, secondary metabolite, and effector coding capacity across Cochliobolus pathogens.

The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25× higher than those between inbred lines and 50× lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP-encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence.