Maize Anthracnose Research Articles

Conserved perception of host and non-host signals via the a-pheromone receptor Ste3 in Colletotrichum graminicola.

Understanding the interactions between fungal plant pathogens and host roots is crucial for developing effective disease management strategies. This study investigates the molecular mechanisms underpinning the chemotropic responses of the maize anthracnose fungus Colletotrichum graminicola to maize root exudates. Combining the generation of a deletion mutant with monitoring of disease symptom development and detailed analysis of chemotropic growth using a 3D-printed device, we identify the 7-transmembrane G-protein coupled receptor (GPCR) CgSte3 as a key player in sensing both plant-derived class III peroxidases and diterpenoids. Activation of CgSte3 initiates signaling through CgSo, a homolog to the Cell Wall Integrity Mitogen-Activated Protein Kinase (CWI MAPK) pathway scaffold protein identified in other filamentous fungi, facilitating the pathogen's growth towards plant defense molecules. The NADPH oxidase CgNox2 is crucial for peroxidase sensing but not for diterpenoid detection. These findings reveal that CgSte3 and CWI MAPK pathways are central to C. graminicola's ability to hijack plant defense signals, highlighting potential targets for controlling maize anthracnose.

Plant immunity suppression by an β-1,3-glucanase of the maize anthracnose pathogen Colletotrichum graminicola

BackgroundMany phytopathogens secrete a large number of cell wall degrading enzymes (CWDEs) to decompose host cell walls in order to penetrate the host, obtain nutrients and accelerate colonization. There is a wide variety of CWDEs produced by plant pathogens, including glycoside hydrolases (GHs), which determine the virulence, pathogenicity, and host specificity of phytopathogens. The specific molecular mechanisms by which pathogens suppress host immunity remain obscure.ResultIn this study, we found that CgEC124 encodes a glycosyl hydrolase with a signal peptide and a conserved Glyco_hydro_cc domain which belongs to glycoside hydrolase 128 family. The expression of CgEC124 was significantly induced in the early stage of Colletotrichum graminicola infection, especially at 12 hpi. Furthermore, CgEC124 positively regulated the pathogenicity, but it did not impact the vegetative growth of mycelia. Ecotopic transient expression of CgEC124 decreased the disease resistance and callose deposition in maize. Moreover, CgEC124 exhibited the β-1,3-glucanase activity and suppresses glucan-induced ROS burst in maize leaves.ConclusionsOur results indicate that CgEC124 is required for full virulence of C. graminicola but not for vegetative growth. CgEC124 increases maize susceptibility by inhibiting host reactive oxygen species burst as well as callose deposition. Meanwhile, our data suggests that CgEC124 explores its β-1,3-glucanase activity to prevent induction of host defenses.

Metabolic re-programming in confrontations of Colletotrichum graminicola and Aspergillus nidulans with Bacillus biocontrol agents

We established confrontations between two different fungi, i.e., the maize anthracnose and stalk rot pathogen Colletotrichum graminicola, and the ubiquitous fungus Aspergillus nidulans, and different biocontrol species, i.e., Bacillus subtilis, Bacillus velezensis, and Bacillus amyloliquefaciens. In all fungus–bacterium confrontations tested, growth arrest and, thus, distance inhibition was observed on solid substrata. LC–MS/MS analyses of culture filtrates suggested formation of several metabolites only synthesized in confrontations. Interestingly, microscopy of fungal hyphae grown in liquid medium showed protrusions and color changes occurred only in media harboring fungus-bacterium confrontations. These observations indicate metabolic re-programming and suggest formation of putative secondary metabolites in interactions involving microbial biocontrol agents.

The galactose metabolism genes UGE1 and UGM1 are novel virulence factors of the maize anthracnose fungus Colletotrichum graminicola.

Fungal cell walls represent the frontline contact with the host and play a prime role in pathogenesis. While the roles of the cell wall polymers like chitin and branched β-glucan are well understood in vegetative and pathogenic development, that of the most prominent galactose-containing polymers galactosaminogalactan and fungal-type galactomannan is unknown in plant pathogenic fungi. Mining the genome of the maize pathogen Colletotrichum graminicola identified the single-copy key galactose metabolism genes UGE1 and UGM1, encoding a UDP-glucose-4-epimerase and UDP-galactopyranose mutase, respectively. UGE1 is thought to be required for biosynthesis of both polymers, whereas UGM1 is specifically required for fungal-type galactomannan formation. Promoter:eGFP fusion strains revealed that both genes are expressed in vegetative and in pathogenic hyphae at all stages of pathogenesis. Targeted deletion of UGE1 and UGM1, and fluorescence-labeling of galactosaminogalactan and fungal-type galactomannan confirmed that Δuge1 mutants were unable to synthesize either of these polymers, and Δugm1 mutants did not exhibit fungal-type galactomannan. Appressoria of Δuge1, but not of Δugm1 mutants, were defective in adhesion, highlighting a function of galactosaminogalactan in the establishment of these infection cells on hydrophobic surfaces. Both Δuge1 and Δugm1 mutants showed cell wall defects in older vegetative hyphae and severely reduced appressorial penetration competence. On intact leaves of Zea mays, both mutants showed strongly reduced disease symptom severity, indicating that UGE1 and UGM1 represent novel virulence factors of C. graminicola.

First Report of Colletotrichum graminicola Causing Maize Anthracnose in Galicia, Northwestern Spain.

Maize (Zea mays) is one of the most important crops worldwide, and fungal diseases are responsible for major losses in food production. Anthracnose caused by Colletotrichum graminicola can infect all maize tissues, although stalk rot and seedling blight cause more significant economic damage (Munkvold and White, 2016). Anthracnose stalk rot is characterized by a distinctive external blackening of the lower stalks resulting in large black streaks, while the pith turns dark brown and has a shredded appearance. Like most stalk rots, the most obvious symptom is a sudden death of plants before grain maturity, and plant lodging. Symptoms commonly appear late in the season, suspicious maize stems of cultivar Tuy exhibiting symptoms of anthracnose stalk rot were collected from a field in Pontevedra, Galicia, Spain (Geographical coordinates: 42°23'27.1" N - 8°30'46.3" W) between June and December of 2022. Stem samples, approximately 50 mm2, were dissected and surface-disinfected for 90 seconds in 20% sodium hypochlorite (v/v) and rinsed three times in sterile distilled water. The samples were transferred to one half-strength acidified potato dextrose agar (PDA) supplemented with ampicillin (100 µg/mL) and lactic acid 90% (1.5 mL/L) and incubated for 5 days at 25 ºC (Sukno et al. 2008). Single spores were transferred to fresh PDA plates to obtain pure culture isolates. A total of six isolates were obtained, and among them, two were selected for further characterization (SP-36820-1 and SP-36820-3). Colonies grown on PDA have dark gray aerial mycelium with orange-colored spore masses. Conidia are falcate, slightly curved, tapered toward the tips, and are produced in acervuli with setae, measuring 37.65 to 24.84 x 8.02 to 4.67 µm, respectively (n = 100). These morphological characteristics are in agreement with C. graminicola previously described by Bergstrom and Nicholson (1999). Isolates were grown in potato dextrose broth (PDB) for 3 days at 25 ºC and total genomic DNA was extracted using a DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA). The internal transcribed spacer region of rDNA and the manganese-type superoxide dismutase gene (SOD2) were amplified using primers ITS4/ITS5 (White et al. 1990) and SOD625/SOD507 (Fang et al. 2002) and consequently sequenced. GenBank BLAST analysis revealed that the sequences were 100% identical to strains of C. graminicola. All sequences were deposited in GenBank (see e-Xtra 1 for accession numbers). To confirm Koch's postulates, plants of a derivative of maize inbred line Mo940 (developmental stage V3) were placed horizontally in a tray for inoculation and 20 droplets (7.5 µL) of a suspension of 3 x 105 conidia per milliliter were placed on the surface of the third leaf. The trays were closed to retain moisture and incubated overnight at 23ºC. The next day, the plants were returned to a vertical position and incubated in a growth chamber at 25ºC with 80% humidity and a light cycle of 16 h of light and 8 h of dark (Vargas et al. 2012). After four days inoculated leaves presented brown elongated lesions with necrotic centers consistent with C. graminicola infection, whereas control plants remained asymptomatic. The strains reisolated from infected leaves were morphologically identical to the original isolates. To our knowledge, this is the first report of Colletotrichum graminicola causing maize anthracnose in Spain. Recently, maize anthracnose was also reported in Bosnia and Herzegovina and China (Duan et al. 2019; Cuevas-Fernández et al. 2019), suggesting the pathogen's geographic range is increasing, which may be a threat to maize cultivation in locations with optimal humid conditions for disease development.

Effector secretion and stability in the maize anthracnose pathogen Colletotrichum graminicola requires N-linked protein glycosylation and the ER chaperone pathway.

N-linked protein glycosylation is a conserved and essential modification mediating protein processing and quality control in the endoplasmic reticulum (ER), but how this contributes to the infection cycle of phytopathogenic fungi is largely unknown. In this study, we discovered that inhibition of protein N-glycosylation severely affected vegetative growth, hyphal tip development, conidial germination, appressorium formation, and, ultimately, the ability of the maize (Zea mays) anthracnose pathogen Colletotrichum graminicola to infect its host. Quantitative proteomics analysis showed that N-glycosylation can coordinate protein O-glycosylation, glycosylphosphatidylinositol anchor modification, and endoplasmic reticulum quality control (ERQC) by directly targeting the proteins from the corresponding pathway in the ER. We performed a functional study of the N-glycosylation pathway-related protein CgALG3 and of the ERQC pathway-related protein CgCNX1, which demonstrated that N-glycosylation of ER chaperone proteins is essential for effector stability, secretion, and pathogenicity of C. graminicola. Our study provides concrete evidence for the regulation of effector protein stability and secretion by N-glycosylation.

Genome Sequence Resource of a Colletotrichum graminicola Field Strain from China.

The maize anthracnose stalk rot and leaf blight diseases caused by the fungal pathogen Colletotrichum graminicola is emerging as an important threat to corn production worldwide. In this work, we provide an improved genome assembly of a C. graminicola strain (TZ-3) by using the PacBio Sequel II and Illumina high-throughput sequencing technologies. The genome of TZ-3 consisting of 36 contigs with a length of 59.3 Mb. By correcting and evaluating with the Illumina sequencing data and BUSCO, this genome showed a high assembly quality and integrity. Gene annotation of this genome predicted 11,911 protein-coding genes, among which 983 secreted protein-coding genes and 332 effector genes were predicted. Comparing with previous genomes of C. graminicola strains, TZ-3 genome is more superior in nearly all parameters. The genome assembly and annotation will enhance our knowledge of the pathogen's genetic makeup and molecular mechanisms underlying its pathogenicity, as well as provide valuable insights into the genome variation across different regions.

Intraspecific variability in growth and in vitro production of plant cell wall-degrading enzymes among argentinean isolates of Colletotrichum graminicola, a maize pathogen

Background and aims: Colletotrichum graminicola (Glomerellaceae, Glomerellales), the causal agent of maize (Zea mays) anthracnose, as many other fungal pathogens, relies on its battery of cell wall degrading enzymes (CWDEs) to make its way through the cell walls of the host, and thus the production of these enzymes is considered a virulence factor. Since this disease is relatively new in Argentina, little is known about the capacity of CWDEs production in native isolates. The aim of this work was to investigate if there is intraspecific variability in growth and in vitro production of several extracellular CWDEs among Argentinean fungal isolates of C. graminicola. M&M: Eight isolates of C. graminicola (obtained from symptomatic maize plants growing in different geographical locations) were tested in vitro to evaluate growth capacity and polygalacturonase, polymethylgalacturonase, β-glucosidase and laccase production, using two different liquid culture media. Results: Polygalacturonase, polymethylgalacturonase and β-glucosidase production greatly varied among isolates and culture media. Laccase activity was detected only in three isolates. Utmost enzymatic titres attained were respectively 250, 280, 45 and 63 U/l. The observed intraspecific variability in CWDEs in vitro production is consistent with the high variability found at genetic level when assessing ISSR markers. Conclusions: The isolates of C. graminicola evaluated showed notable differences in CWDEs production, not associated with a differential growth. This indicates a large intraspecific variability, which might be considered when choosing a method to deal with this pathogen.

Population Genomics Provide Insights into the Global Genetic Structure of Colletotrichum graminicola, the Causal Agent of Maize Anthracnose.

Understanding the genetic diversity and mechanisms underlying genetic variation in pathogen populations is crucial to the development of effective control strategies. We investigated the genetic diversity and reproductive biology of Colletotrichum graminicola isolates which infect maize by sequencing the genomes of 108 isolates collected from 14 countries using restriction site-associated DNA sequencing (RAD-seq) and whole-genome sequencing (WGS). Clustering analyses based on single-nucleotide polymorphisms revealed three genetic groups delimited by continental origin, compatible with short-dispersal of the pathogen and geographic subdivision. Intra- and intercontinental migration was observed between Europe and South America, likely associated with the movement of contaminated germplasm. Low clonality, evidence of genetic recombination, and high phenotypic diversity were detected. We show evidence that, although it is rare (possibly due to losses of sexual reproduction- and meiosis-associated genes) C. graminicola can undergo sexual recombination. Our results support the hypotheses that intra- and intercontinental pathogen migration and genetic recombination have great impacts on the C. graminicola population structure. IMPORTANCE Plant pathogens cause significant reductions in yield and crop quality and cause enormous economic losses worldwide. Reducing these losses provides an obvious strategy to increase food production without further degrading natural ecosystems; however, this requires knowledge of the biology and evolution of the pathogens in agroecosystems. We employed a population genomics approach to investigate the genetic diversity and reproductive biology of the maize anthracnose pathogen (Colletotrichum graminicola) in 14 countries. We found that the populations are correlated with their geographical origin and that migration between countries is ongoing, possibly caused by the movement of infected plant material. This result has direct implications for disease management because migration can cause the movement of more virulent and/or fungicide-resistant genotypes. We conclude that genetic recombination is frequent (in contrast to the traditional view of C. graminicola being mainly asexual), which strongly impacts control measures and breeding programs aimed at controlling this disease.

The Small Ras Superfamily GTPase Rho4 of the Maize Anthracnose Fungus Colletotrichum graminicola Is Required for β-1,3-glucan Synthesis, Cell Wall Integrity, and Full Virulence.

Small Ras superfamily GTPases are highly conserved regulatory factors of fungal cell wall biosynthesis and morphogenesis. Previous experiments have shown that the Rho4-like protein of the maize anthracnose fungus Colletotrichum graminicola, formerly erroneously annotated as a Rho1 protein, physically interacts with the β-1,3-glucan synthase Gls1 (Lange et al., 2014; Curr. Genet. 60:343–350). Here, we show that Rho4 is required for β-1,3-glucan synthesis. Accordingly, Δrho4 strains formed distorted vegetative hyphae with swellings, and exhibited strongly reduced rates of hyphal growth and defects in asexual sporulation. Moreover, on host cuticles, conidia of Δrho4 strains formed long hyphae with hyphopodia, rather than short germ tubes with appressoria. Hyphopodia of Δrho4 strains exhibited penetration defects and often germinated laterally, indicative of cell wall weaknesses. In planta differentiated infection hyphae of Δrho4 strains were fringy, and anthracnose disease symptoms caused by these strains on intact and wounded maize leaf segments were significantly weaker than those caused by the WT strain. A retarded disease symptom development was confirmed by qPCR analyses. Collectively, we identified the Ras GTPase Rho4 as a new virulence factor of C. graminicola.

Maize Anthracnose Stalk Rot in the Genomic Era.

Anthracnose stalk rot (ASR) of maize results in millions of dollars in losses annually in the United States. ASR, together with anthracnose leaf blight and anthracnose top dieback, is caused by the fungus Colletotrichum graminicola. Current ASR management recommendations emphasize host resistance and reduction of plant stressors (e.g., drought, heat, low fertility, or soil acidity). Stress reduction may be more difficult to achieve in the future due to more high-intensity production protocols and climate change. Moreover, cultural and chemical management practices may conflict with other important goals, including environmental sustainability and maximization of yield potential. Thus, future ASR management may rely more heavily on host resistance, for which there are relatively few highly effective sources. The last comprehensive review of C. graminicola and maize anthracnose was written over two decades ago. The genomic age has brought important new insights into mechanisms governing the host-pathogen interaction from the application of molecular and cytological technologies. This review provides a summary of our current model of maize anthracnose etiology, including how increased knowledge of molecular and cellular events could contribute to better ASR management. Improved understanding of C. graminicola taxonomy has confirmed that the fungus is specific to Zea mays, and that it colonizes living maize tissues via a critical biotrophic phase. Successful biotrophic establishment relies on an array of secreted protein effectors and secondary metabolites produced at different stages of infection and dispersed to multiple locations. These molecules could provide therapeutic targets for the next generation of transgenic or gene-edited ASR-resistant hybrids.

Practical Applications of China A-Shares: Strategic Allocation to Market and Factor Premiums

<h3>Abstract</h3> <h3>Background</h3> <i>Colletotrichum graminicola</i>, the causal agent of maize anthracnose, is an important crop disease worldwide. Understanding the genetic diversity and mechanisms underlying genetic variation in pathogen populations is crucial to the development of effective control strategies. The genus <i>Colletotrichum</i> is largely recognized as asexual, but several species have been reported to have a sexual cycle. Here, we employed a population genomics approach to investigate the genetic diversity and reproductive biology of <i>C. graminicola</i> isolates infecting maize. We sequenced 108 isolates of <i>C. graminicola</i> collected in 14 countries using restriction site-associated DNA sequencing (RAD-Seq) and whole-genome sequencing (WGS). <h3>Results</h3> Clustering analyses based on single-nucleotide polymorphisms showed populational differentiation at a global scale, with three genetic groups delimited by continental origin, compatible with short-dispersal of the pathogen, and geographic subdivision. Distinct levels of genetic diversity were observed between these clades, suggesting different evolutionary histories. Intra and inter-continental migration was predicted between Europe and South America, likely associated with the movement of contaminated germplasm. Low clonality and evidence of genetic recombination were detected from the analysis of linkage disequilibrium and the pairwise homoplasy index (PHI) test for clonality. We show evidence that even if rare (possibly due to losses of sex and meiosis-associated genes) <i>C. graminicola</i> can undergo sexual recombination based on lab assays and genomic analyses. <h3>Conclusions</h3> Our results support hypotheses of intra and intercontinental pathogen migration and genetic recombination with great impact on <i>C. graminicola</i> population structure.

Molecular Characterization of the Purine Degradation Pathway Genes ALA1 and URE1 of the Maize Anthracnose Fungus Colletotrichum graminicola Identified Urease as a Novel Target for Plant Disease Control.

Fungal pathogenicity is governed by environmental factors, with nitrogen playing a key role in triggering pathogenic development. Spores germinating on the plant cuticle are exposed to a nitrogen-free environment, and reprograming of nitrogen metabolism is required for bridging the time needed to gain access to the nitrogen sources of the host. Although degradation of endogenous purine bases efficiently generates ammonium and may allow the fungus to bridge the preinvasion nitrogen gap, the roles of the purine degradation pathway and of the key genes encoding allantoicase and urease are largely unknown in plant pathogenic fungi. To investigate the roles of the allantoicase and urease genes ALA1 and URE1 of the maize anthracnose fungus Colletotrichum graminicola in pathogenic development, we generated ALA1:eGFP and URE1:eGFP fusion strains as well as allantoicase- and urease-deficient mutants. Virulence assays, live cell, and differential interference contrast imaging, chemical complementation and employment of a urease inhibitor showed that the purine degradation genes ALA1 and URE1 are required for bridging nitrogen deficiency at early phases of the infection process and for full virulence. Application of the urease inhibitor acetohydroxamic acid did not only protect maize from C. graminicola infection, but also interfered with the infection process of the wheat powdery mildew fungus Blumeria graminis f. sp. tritici, the maize and broad bean rusts Puccinia sorghi and Uromyces viciae-fabae, and the potato late blight pathogen Phytophthora infestans. Our data strongly suggest that inhibition of the purine degradation pathway might represent a novel approach to control plant pathogenic fungi and oomycetes.

Bioinformatic analysis and functional characterization of the CFEM proteins in maize anthracnose fungus Colletotrichum graminicola

Fungal secreted proteins that contain the Common in Fungal Extracellular Membrane (CFEM) domain are important for pathogenicity. The hemibiotrophic fungus Colletotrichum graminicola causes the serious anthracnose disease of maize. In this study, we identified 24 CgCFEM proteins in the genome of C. graminicola. Phylogenic analysis revealed that these 24 proteins (CgCFEM1—24) can be divided into 2 clades based on the presence of the trans-membrane domain. Sequence alignment analysis indicated that the amino acids of the CFEM domain are highly conserved and contain 8 spaced cysteines, with the exception that CgCFEM1 and CgCFEM24 lack 1 and 2 cysteines, respectively. Ten CgCFEM proteins with a signal peptide and without the trans-membrane domain were considered as candidate effectors and, thus were selected for structural prediction and functional analyses. The CFEM domain in the candidate effectors can form a helical-basket structure homologous to the Csa2 protein in Candida albicans, which is responsible for haem acquisition and pathogenicity. Subcellular localization analysis revealed that these effectors accumulate in the cell membrane, nucleus, and cytosolic bodies. Additionally, 5 effectors, CgCFEM6, 7, 8, 9 and 15, can suppress the BAX-induced programmed cell death in Nicotiana benthamiana with or without the signal peptide. These results demonstrate that these 10 CgCFEM candidate effectors with different structures and subcellular localizations in host cells may play important roles during the pathogenic processes on maize plants.

Two genes in a pathogenicity gene cluster encoding secreted proteins are required for appressorial penetration and infection of the maize anthracnose fungus Colletotrichum graminicola.

To avoid pathogen-associated molecular pattern recognition, the hemibiotrophic maize pathogen Colletotrichum graminicola secretes proteins mediating the establishment of biotrophy. Targeted deletion of 26 individual candidate genes and seven gene clusters comprising 32 genes of C. graminicola identified a pathogenicity cluster (CLU5) of five co-linear genes, all of which, with the exception of CLU5b, encode secreted proteins. Targeted deletion of all genes of CLU5 revealed that CLU5a and CLU5d are required for full appressorial penetration competence, with virulence deficiencies independent of the host genotype and organ inoculated. Cytorrhysis experiments and microscopy showed that Δclu5a mutants form pressurized appressoria, but they are hampered in forming penetration pores and fail to differentiate a penetration peg. Whereas Δclu5d mutants elicited WT-like papillae, albeit at increased frequencies, papillae induced by Δclu5a mutants were much smaller than those elicited by the WT. Synteny of CLU5 is not only conserved in Colletotrichum spp. but also in additional species of Sordariomycetes including insect pathogens and saprophytes suggesting importance of CLU5 for fungal biology. Since CLU5a and CLU5d also occur in non-pathogenic fungi and since they are expressed prior to plant invasion and even in vegetative hyphae, the encoded proteins probably do not act primarily as effectors.

The Glycosylphosphatidylinositol Anchor Biosynthesis Genes GPI12, GAA1, and GPI8 Are Essential for Cell-Wall Integrity and Pathogenicity of the Maize Anthracnose Fungus Colletotrichum graminicola.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is one of the most common posttranslational modifications of proteins in eukaryotic cells and is important for associating proteins with the cell surface. In fungi, GPI-anchored proteins play essential roles in cross-linking of β-glucan cell-wall polymers and cell-wall rigidity. GPI-anchor synthesis is successively performed at the cytoplasmic and the luminal face of the ER membrane and involves approximately 25 proteins. While mutagenesis of auxiliary genes of this pathway suggested roles of GPI-anchored proteins in hyphal growth and virulence, essential genes of this pathway have not been characterized. Taking advantage of RNA interference (RNAi) we analyzed the function of the three essential genes GPI12, GAA1 and GPI8, encoding a cytoplasmic N-acetylglucosaminylphosphatidylinositol deacetylase, a metallo-peptide-synthetase and a cystein protease, the latter two representing catalytic components of the GPI transamidase complex. RNAi strains showed drastic cell-wall defects, resulting in exploding infection cells on the plant surface and severe distortion of in planta-differentiated infection hyphae, including formation of intrahyphal hyphae. Reduction of transcript abundance of the genes analyzed resulted in nonpathogenicity. We show here for the first time that the GPI synthesis genes GPI12, GAA1, and GPI8 are indispensable for vegetative development and pathogenicity of the causal agent of maize anthracnose, Colletotrichum graminicola.

A highly conserved metalloprotease effector enhances virulence in the maize anthracnose fungus Colletotrichum graminicola.

Colletotrichum graminicola causes maize anthracnose, an agronomically important disease with a worldwide distribution. We have identified a fungalysin metalloprotease (Cgfl) with a role in virulence. Transcriptional profiling experiments and live cell imaging show that Cgfl is specifically expressed during the biotrophic stage of infection. To determine whether Cgfl has a role in virulence, we obtained null mutants lacking Cgfl and performed pathogenicity and live microscopy assays. The appressorium morphology of the null mutants is normal, but they exhibit delayed development during the infection process on maize leaves and roots, showing that Cgfl has a role in virulence. In vitro chitinase activity assays of leaves infected with wild-type and null mutant strains show that, in the absence of Cgfl, maize leaves exhibit increased chitinase activity. Phylogenetic analyses show that Cgfl is highly conserved in fungi. Similarity searches, phylogenetic analysis and transcriptional profiling show that C. graminicola encodes two LysM domain-containing homologues of Ecp6, suggesting that this fungus employs both Cgfl-mediated and LysM protein-mediated strategies to control chitin signalling.

A Colletotrichum graminicola mutant deficient in the establishment of biotrophy reveals early transcriptional events in the maize anthracnose disease interaction.

BackgroundColletotrichum graminicola is a hemibiotrophic fungal pathogen that causes maize anthracnose disease. It progresses through three recognizable phases of pathogenic development in planta: melanized appressoria on the host surface prior to penetration; biotrophy, characterized by intracellular colonization of living host cells; and necrotrophy, characterized by host cell death and symptom development. A “Mixed Effects” Generalized Linear Model (GLM) was developed and applied to an existing Illumina transcriptome dataset, substantially increasing the statistical power of the analysis of C. graminicola gene expression during infection and colonization. Additionally, the in planta transcriptome of the wild-type was compared with that of a mutant strain impaired in the establishment of biotrophy, allowing detailed dissection of events occurring specifically during penetration, and during early versus late biotrophy.ResultsMore than 2000 fungal genes were differentially transcribed during appressorial maturation, penetration, and colonization. Secreted proteins, secondary metabolism genes, and membrane receptors were over-represented among the differentially expressed genes, suggesting that the fungus engages in an intimate and dynamic conversation with the host, beginning prior to penetration. This communication process probably involves reception of plant signals triggering subsequent developmental progress in the fungus, as well as production of signals that induce responses in the host. Later phases of biotrophy were more similar to necrotrophy, with increased production of secreted proteases, inducers of plant cell death, hydrolases, and membrane bound transporters for the uptake and egress of potential toxins, signals, and nutrients.ConclusionsThis approach revealed, in unprecedented detail, fungal genes specifically expressed during critical phases of host penetration and biotrophic establishment. Many encoded secreted proteins, secondary metabolism enzymes, and receptors that may play roles in host-pathogen communication necessary to promote susceptibility, and thus may provide targets for chemical or biological controls to manage this important disease. The differentially expressed genes could be used as ‘landmarks’ to more accurately identify developmental progress in compatible versus incompatible interactions involving genetic variants of both host and pathogen.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2546-0) contains supplementary material, which is available to authorized users.

A Fungal Effector With Host Nuclear Localization and DNA-Binding Properties Is Required for Maize Anthracnose Development.

Plant pathogens have the capacity to manipulate the host immune system through the secretion of effectors. We identified 27 putative effector proteins encoded in the genome of the maize anthracnose pathogen Colletotrichum graminicola that are likely to target the host's nucleus, as they simultaneously contain sequence signatures for secretion and nuclear localization. We functionally characterized one protein, identified as CgEP1. This protein is synthesized during the early stages of disease development and is necessary for anthracnose development in maize leaves, stems, and roots. Genetic, molecular, and biochemical studies confirmed that this effector targets the host's nucleus and defines a novel class of double-stranded DNA-binding protein. We show that CgEP1 arose from a gene duplication in an ancestor of a lineage of monocot-infecting Colletotrichum spp. and has undergone an intense evolution process, with evidence for episodes of positive selection. We detected CgEP1 homologs in several species of a grass-infecting lineage of Colletotrichum spp., suggesting that its function may be conserved across a large number of anthracnose pathogens. Our results demonstrate that effectors targeted to the host nucleus may be key elements for disease development and aid in the understanding of the genetic basis of anthracnose development in maize plants.

Root inoculation with Pseudomonas putida KT2440 induces transcriptional and metabolic changes and systemic resistance in maize plants.

Pseudomonas putida KT2440 (KT2440) rhizobacteria colonize a wide range of plants. They have been extensively studied for their capacity to adhere to maize seeds, to tolerate toxic secondary metabolites produced by maize roots and to be attracted by maize roots. However, the response of maize plants to KT2440 colonization has not been investigated yet. Maize roots were inoculated with KT2440 and the local (roots) and systemic (leaves) early plant responses were investigated. The colonization behavior of KT2440 following application to maize seedlings was investigated and transcriptional analysis of stress- and defense-related genes as well as metabolite profiling of local and systemic maize tissues of KT2440-inoculated were performed. The local and systemic responses differed and more pronounced changes were observed in roots compared to leaves. Early in the interaction roots responded via jasmonic acid- and abscisic acid-dependent signaling. Interestingly, during later steps, the salicylic acid pathway was suppressed. Metabolite profiling revealed the importance of plant phospholipids in KT2440-maize interactions. An additional important maize secondary metabolite, a form of benzoxazinone, was also found to be differently abundant in roots 3 days after KT2440 inoculation. However, the transcriptional and metabolic changes observed in bacterized plants early during the interaction were minor and became even less pronounced with time, indicating an accommodation state of the plant to the presence of KT2440. Since the maize plants reacted to the presence of KT2440 in the rhizosphere, we also investigated the ability of these bacteria to trigger induced systemic resistance (ISR) against the maize anthracnose fungus Colletotrichum graminicola. The observed resistance was expressed as strongly reduced leaf necrosis and fungal growth in infected bacterized plants compared to non-bacterized controls, showing the potential of KT2440 to act as resistance inducers.