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 25x higher than those between inbred lines and 50x 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

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

The Role of Mitogen-Activated Protein (MAP) Kinase Signaling Components in the Fungal Development, Stress Response and Virulence of the Fungal Cereal Pathogen Bipolaris sorokiniana.

Mitogen-activated protein kinases (MAPKs) have been demonstrated to be involved in fungal development, sexual reproduction, pathogenicity and/or virulence in many filamentous plant pathogenic fungi, but genes for MAPKs in the fungal cereal pathogen Bipolaris sorokiniana have not been characterized. In this study, orthologues of three MAPK genes (CsSLT2, CsHOG1 and CsFUS3) and one MAPK kinase kinase (MAPKKK) gene (CsSTE11) were identified in the whole genome sequence of the B. sorokiniana isolate ND90Pr, and knockout mutants were generated for each of them. The ∆Csfus3 and ∆Csste11 mutants were defective in conidiation and formation of appressoria-like structures, showed hypersensitivity to oxidative stress and lost pathogenicity on non-wounded leaves of barley cv. Bowman. When inoculated on wounded leaves of Bowman, the ∆Csfus3 and ∆Csste11 mutants were reduced in virulence compared to the wild type. No morphological changes were observed in the ∆Cshog1 mutants in comparison with the wild type; however, they were slightly reduced in growth under oxidative stress and were hypersensitive to hyperosmotic stress. The ∆Cshog1 mutants formed normal appressoria-like structures but were reduced in virulence when inoculated on Bowman leaves. The ∆Csslt2 mutants produced more vegetative hyphae, had lighter pigmentation, were more sensitive to cell wall degrading enzymes, and were reduced in virulence on Bowman leaves, although they formed normal appressoria like the wild type. Root infection assays indicated that the ∆Cshog1 and ∆Csslt2 mutants were able to infect barley roots while the ∆Csfus3 and ∆Csste11 failed to cause any symptoms. However, no significant difference in virulence was observed for ∆Cshog1 mutants while ∆Csslt2 mutants showed significantly reduced virulence on barley roots in comparison with the wild type. Our results indicated that all of these MAPK and MAPKKK genes are involved in the regulation of fungal development under normal and stress conditions and required for full virulence on barley plants

Primers used in this study.

<p>*Underlined sequences are complementary to M13F and M13R sequences, respectively.</p><p>Primers used in this study.</p

Fungal development and conidial productivity of knockout mutants.

<p>A, fungal growth and responses to stresses. Colonies were grown on PDA, PDA with 10mM H₂O₂, PDA with 1 M KCl and PDA with 1.5 M sorbitol, respectively, starting with mycelial plugs of uniform size inoculated on the centers of the plates. Photos were taken after growth for 6 days at 25°C. B, Quantitative growth rate of wild-type and mutants grown on PDA, PDA with 1 M KCl, PDA with 1.5 M sorbitol and PDA with 10mM H₂O₂, respectively. The colony diameters were measured 6 days after growth on the media. Error bars indicate standard deviation. C, Conidial productivity of wild-type and mutants grown on minimal medium plates for 6 days at 25°C in a cycle of 14 h of light and 10 h of darkness. Error bars indicate standard deviation. Significant differences (P value = 0.001) are indicated by different letters above the columns under each condition.</p

Pathogenicity test on barley roots.

<p>Roots (15cm long) of barley seedlings (cv. Bowman) were inoculated with mycelial plugs (2×2 mm²) of wild type (ND90Pr) and mutants according to the method described by Liljeroth et al. (23). The length of brown discoloration lesions was measured at 9 days after inoculation. Error bars indicate standard deviation. Significant differences (P value = 0.001) are indicated by different letters above the columns.</p

Infection structure differentiation of the wild type (ND90Pr) (A), <i>∆Csfus3-1</i> (B), <i>∆Csste11-1</i> (C), <i>∆Cshog1-4</i> (D) and <i>∆Csslt2-10</i> (E) strains at 24 hours after inoculation (HAI) on intact leaves of barley cv.

<p><b>Bowman.</b> Fungal and dead plant cells were stained blue with trypan blue. Appressoria are indicated by black arrows. Bars = 60 μm.</p

Pathogenicity test of mutants on barley leaves.

<p>A, Point-inoculation with mycelial plugs of wild type (ND90Pr), <i>∆Csfus3-1</i> and <i>∆Csste11-1</i> mutants on non-wounded (Wt) and wounded (W) leaves of barley cv. Bowman. B, Spray inoculation of conidia of wild type (ND90Pr), <i>∆Cshog1-4</i> and <i>∆Csslt2-10</i> mutants on intact leaves of barley cv. Bowman. C. Quantitative analysis of disease severity based on lesion lengths on inoculated leaves as shown in A. All photographs were taken at 5 days after inoculation (DAI). A syringe needle was used to make wounds on leaves for inoculation with small mycelial plugs (A). Error bars indicate standard deviation. Significant differences (P value = 0.001) are indicated by different letters above the columns.</p

Generation of ∆<i>Cshog1</i> strains of <i>Bipolaris sorokiniana</i>.

<p>A, a diagram showing replacement of the <i>CsHOG1</i> gene by a 2.6 kb fragment carrying the <i>E</i>. <i>coli</i> hygromycin phosphotransferase gene (<i>hph</i>) using the split-marker system [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128291#pone.0128291.ref017" target="_blank">17</a>]. B, Southern hybridization of <i>Xba</i> I-digested genomic DNA from the wild type and ∆<i>Cshog1</i> strains using probe amplified with primers CsHOG1-F1+CsHOG1-F2. The 1.7 kb fragment in the wild type strain (ND90Pr) was replaced by the 3.3 kb fragment in the ∆<i>Cshog1</i> strains (∆<i>Cshog1</i>-2 and ∆<i>Cshog1</i>-4). <i>ect</i>-<i>Cshog1</i>-3 is an ectopic transformant.</p

RNA-Seq Revealed Differences in Transcriptomes between 3ADON and 15ADON Populations of Fusarium graminearum In Vitro and In Planta.

Fusarium graminearum is the major causal agent of Fusarium head blight (FHB) in barley and wheat in North America. The fungus not only causes yield loss of the crops but also produces harmful trichothecene mycotoxins [Deoxynivalenol (DON) and its derivatives-3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON), and nivalenol (NIV)] that contaminate grains. Previous studies showed a dramatic increase of 3ADON-producing isolates with higher aggressiveness and DON production than the 15ADON-producing isolates in North America. However, the genetic and molecular basis of differences between the two types of isolates is unclear. In this study, we compared transcriptomes of the 3ADON and 15ADON isolates in vitro (in culture media) and in planta (during infection on the susceptible wheat cultivar 'Briggs') using RNA-sequencing. The in vitro gene expression comparison identified 479 up-regulated and 801 down-regulated genes in the 3ADON isolates; the up-regulated genes were mainly involved in C-compound and carbohydrate metabolism (18.6%), polysaccharide metabolism (7.7%) or were of unknown functions (57.6%). The in planta gene expression analysis revealed that 185, 89, and 62 genes were up-regulated in the 3ADON population at 48, 96, and 144 hours after inoculation (HAI), respectively. The up-regulated genes were significantly enriched in functions for cellular import, C-compound and carbohydrate metabolism, allantoin and allantoate transport at 48 HAI, for detoxification and virulence at 96 HAI, and for metabolism of acetic acid derivatives, detoxification, and cellular import at 144 HAI. Comparative analyses of in planta versus in vitro gene expression further revealed 2,159, 1,981 and 2,095 genes up-regulated in the 3ADON isolates, and 2,415, 2,059 and 1,777 genes up-regulated in the 15ADON isolates at the three time points after inoculation. Collectively, our data provides a foundation for further understanding of molecular mechanisms involved in aggressiveness and DON production of the two chemotype isolates of F. graminearum