Evolutionary history of the cytochrome P450s from Colletotrichum species and prediction of their putative functional roles during host-pathogen interactions.

The genomes of species belonging to the genus Colletotrichum harbor a substantial number of cytochrome P450 monooxygenases (CYPs) encoded by a broad diversity of gene families. However, the biological role of their CYP complement (CYPome) has not been elucidated. Here, we investigated the putative evolutionary scenarios that occurred during the evolution of the CYPome belonging to the Colletotrichum Graminicola species complex (s.c.) and their biological implications. The study revealed that most of the CYPome gene families belonging to the Graminicola s.c. experienced gene contractions. The reductive evolution resulted in species restricted CYPs are predominant in each CYPome of members from the Graminicola s.c., whereas only 18 families are absolutely conserved among these species. However, members of CYP families displayed a notably different phylogenetic relationship at the tertiary structure level, suggesting a putative convergent evolution scenario. Most of the CYP enzymes of the Graminicola s.c. share redundant functions in secondary metabolite biosynthesis and xenobiotic metabolism. Hence, this current work suggests that the presence of a broad CYPome in the genus Colletotrichum plays a critical role in the optimization of the colonization capability and virulence.

Chromosome-level analysis of the Colletotrichum graminicola genome reveals the unique characteristics of core and minichromosomes.

The fungal pathogen Colletotrichum graminicola causes the anthracnose of maize (Zea mays) and is responsible for significant yield losses worldwide. The genome of C. graminicola was sequenced in 2012 using Sanger sequencing, 454 pyrosequencing, and an optical map to obtain an assembly of 13 pseudochromosomes. We re-sequenced the genome using a combination of short-read (Illumina) and long-read (PacBio) technologies to obtain a chromosome-level assembly. The new version of the genome sequence has 13 chromosomes with a total length of 57.43 Mb. We detected 66 (23.62 Mb) structural rearrangements in the new assembly with respect to the previous version, consisting of 61 (21.98 Mb) translocations, 1 (1.41 Mb) inversion, and 4 (221 Kb) duplications. We annotated the genome and obtained 15,118 predicted genes and 3,614 new gene models compared to the previous version of the assembly. We show that 25.88% of the new assembly is composed of repetitive DNA elements (13.68% more than the previous assembly version), which are mostly found in gene-sparse regions. We describe genomic compartmentalization consisting of repeat-rich and gene-poor regions vs. repeat-poor and gene-rich regions. A total of 1,140 secreted proteins were found mainly in repeat-rich regions. We also found that ~75% of the three smallest chromosomes (minichromosomes, between 730 and 551 Kb) are strongly affected by repeat-induced point mutation (RIP) compared with 28% of the larger chromosomes. The gene content of the minichromosomes (MCs) comprises 121 genes, of which 83.6% are hypothetical proteins with no predicted function, while the mean percentage of Chr1-Chr10 is 36.5%. No predicted secreted proteins are present in the MCs. Interestingly, only 2% of the genes in Chr11 have homologs in other strains of C. graminicola, while Chr12 and 13 have 58 and 57%, respectively, raising the question as to whether Chrs12 and 13 are dispensable. The core chromosomes (Chr1-Chr10) are very different with respect to the MCs (Chr11-Chr13) in terms of the content and sequence features. We hypothesize that the higher density of repetitive elements and RIPs in the MCs may be linked to the adaptation and/or host co-evolution of this pathogenic fungus.

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.

Comparative transcriptomic provides novel insights into the soybean response to Colletotrichum truncatum infection.

Introduction: Soybean (Glycine max) is among the most important crops in the world, and its production can be threatened by biotic diseases, such as anthracnose. Soybean anthracnose is a seed-borne disease mainly caused by the hemibiotrophic fungus Colletotrichum truncatum. Typical symptoms are pre- and post-emergence damping off and necrotic lesions on cotyledons, petioles, leaves, and pods. Anthracnose symptoms can appear early in the field, causing major losses to soybean production. Material and Methods: In preliminary experiments, we observed that the same soybean cultivar can have a range of susceptibility towards different strains of C. truncatum, while the same C. truncatum strain can cause varying levels of disease severity in different soybean cultivars. To gain a better understanding of the molecular mechanisms regulating the early response of different soybean cultivars to different C. truncatum strains, we performed pathogenicity assays to select two soybean cultivars with significantly different susceptibility to two different C. truncatum strains and analyzed their transcriptome profiles at different time points of interaction (0, 12, 48, and 120 h post-inoculation, hpi). Results and Discussion: The pathogenicity assays showed that the soybean cultivar Gm1 is more resistant to C. truncatum strain 1080, and it is highly susceptible to strain 1059, while cultivar Gm2 shows the opposite behavior. However, if only trivial anthracnose symptoms appeared in the more resistant phenotype (MRP; Gm1-1080; Gm2-1059) upon 120 hpi, in the more susceptible phenotype (MSP; Gm-1059; Gm2- 1080) plants show mild symptoms already at 72 hpi, after which the disease evolved rapidly to severe necrosis and plant death. Interestingly, several genes related to different cellular responses of the plant immune system (pathogen recognition, signaling events, transcriptional reprogramming, and defense-related genes) were commonly modulated at the same time points only in both MRP. The list of differentially expressed genes (DEGs) specific to the more resistant combinations and related to different cellular responses of the plant immune system may shed light on the important host defense pathways against soybean anthracnose.

Nitric Oxide Metabolism Affects Germination in Botrytiscinerea and Is Connected to Nitrate Assimilation.

Nitric oxide regulates numerous physiological processes in species from all taxonomic groups. Here, its role in the early developmental stages of the fungal necrotroph Botrytis cinerea was investigated. Pharmacological analysis demonstrated that NO modulated germination, germ tube elongation and nuclear division rate. Experimental evidence indicates that exogenous NO exerts an immediate but transitory negative effect, slowing down germination-associated processes, and that this effect is largely dependent on the flavohemoglobin BCFHG1. The fungus exhibited a "biphasic response" to NO, being more sensitive to low and high concentrations than to intermediate levels of the NO donor. Global gene expression analysis in the wild-type and ΔBcfhg1 strains indicated a situation of strong nitrosative and oxidative stress determined by exogenous NO, which was much more intense in the mutant strain, that the cells tried to alleviate by upregulating several defense mechanisms, including the simultaneous upregulation of the genes encoding the flavohemoglobin BCFHG1, a nitronate monooxygenase (NMO) and a cyanide hydratase. Genetic evidence suggests the coordinated expression of Bcfhg1 and the NMO coding gene, both adjacent and divergently arranged, in response to NO. Nitrate assimilation genes were upregulated upon exposure to NO, and BCFHG1 appeared to be the main enzymatic system involved in the generation of the signal triggering their induction. Comparative expression analysis also showed the influence of NO on other cellular processes, such as mitochondrial respiration or primary and secondary metabolism, whose response could have been mediated by NmrA-like domain proteins.

A Major Effect Gene Controlling Development and Pathogenicity in Botrytis cinerea Identified Through Genetic Analysis of Natural Mycelial Non-pathogenic Isolates.

Botrytis cinerea is a necrotrophic plant pathogenic fungus with a wide host range. Its natural populations are phenotypically and genetically very diverse. A survey of B. cinerea isolates causing gray mold in the vineyards of Castilla y León, Spain, was carried out and as a result eight non-pathogenic natural variants were identified. Phenotypically these isolates belong to two groups. The first group consists of seven isolates displaying a characteristic mycelial morphotype, which do not sporulate and is unable to produce sclerotia. The second group includes one isolate, which sporulates profusely and does not produce sclerotia. All of them are unresponsive to light. Crosses between a representative mycelial non-pathogenic isolate and a highly aggressive field isolate revealed that the phenotypic differences regarding pathogenicity, sporulation and production of sclerotia cosegregated in the progeny and are determined by a single genetic locus. By applying a bulked segregant analysis strategy based on the comparison of the two parental genomes the locus was mapped to a 110 kb region in chromosome 4. Subcloning and transformation experiments revealed that the polymorphism is an SNP affecting gene Bcin04g03490 in the reference genome of B. cinerea. Genetic complementation analysis and sequencing of the Bcin04g03490 alleles demonstrated that the mutations in the mycelial isolates are allelic and informed about the nature of the alterations causing the phenotypes observed. Integration of the allele of the pathogenic isolate into the non-pathogenic isolate fully restored the ability to infect, to sporulate and to produce sclerotia. Therefore, it is concluded that a major effect gene controlling differentiation and developmental processes as well as pathogenicity has been identified in B. cinerea. It encodes a protein with a GAL4-like Zn(II)2Cys6 binuclear cluster DNA binding domain and an acetyltransferase domain, suggesting a role in regulation of gene expression through a mechanism involving acetylation of specific substrates.