Shaobin Zhong

Obligate biotrophy features unraveled by the genomic analysis of rust fungi

Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance or suppression of plant innate immunity. We sequenced the 101-Mb genome of Melampsora larici-populina, the causal agent of poplar leaf rust, and the 89-Mb genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,399 predicted proteins of M. larici-populina with the 17,773 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic lifestyle include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins, impaired nitrogen and sulfur assimilation pathways, and expanded families of amino acid and oligopeptide membrane transporters. The dramatic up-regulation of transcripts coding for small secreted proteins, secreted hydrolytic enzymes, and transporters in planta suggests that they play a role in host infection and nutrient acquisition. Some of these genomic hallmarks are mirrored in the genomes of other microbial eukaryotes that have independently evolved to infect plants, indicating convergent adaptation to a biotrophic existence inside plant cells.

Obligate Biotrophy Features Unraveled by the Genomic Analysis of the Rust Fungi, Melampsora larici-populina and Puccinia graminis f. sp. tritici

Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance or suppression of plant innate immunity. We sequenced the 101-Mb genome of Melampsora larici-populina, the causal agent of poplar leaf rust, and the 89-Mb genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,399 predicted proteins of M. larici-populina with the 17,773 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic lifestyle include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins, impaired nitrogen and sulfur assimilation pathways, and expanded families of amino acid and oligopeptide membrane transporters. The dramatic up-regulation of transcripts coding for small secreted proteins, secreted hydrolytic enzymes, and transporters in planta suggests that they play a role in host infection and nutrient acquisition. Some of these genomic hallmarks are mirrored in the genomes of other microbial eukaryotes that have independently evolved to infect plants, indicating convergent adaptation to a biotrophic existence inside plant cells.

Diverse Lifestyles and Strategies of Plant Pathogenesis Encoded in the Genomes of Eighteen Dothideomycetes Fungi

The class Dothideomycetes is one of the largest groups of fungi with a high level of ecological diversity including many plant pathogens infecting a broad range of hosts. Here, we compare genome features of 18 members of this class, including 6 necrotrophs, 9 (hemi)biotrophs and 3 saprotrophs, to analyze genome structure, evolution, and the diverse strategies of pathogenesis. The Dothideomycetes most likely evolved from a common ancestor more than 280 million years ago. The 18 genome sequences differ dramatically in size due to variation in repetitive content, but show much less variation in number of (core) genes. Gene order appears to have been rearranged mostly within chromosomal boundaries by multiple inversions, in extant genomes frequently demarcated by adjacent simple repeats. Several Dothideomycetes contain one or more gene-poor, transposable element (TE)-rich putatively dispensable chromosomes of unknown function. The 18 Dothideomycetes offer an extensive catalogue of genes involved in cellulose degradation, proteolysis, secondary metabolism, and cysteine-rich small secreted proteins. Ancestors of the two major orders of plant pathogens in the Dothideomycetes, the Capnodiales and Pleosporales, may have had different modes of pathogenesis, with the former having fewer of these genes than the latter. Many of these genes are enriched in proximity to transposable elements, suggesting faster evolution because of the effects of repeat induced point (RIP) mutations. A syntenic block of genes, including oxidoreductases, is conserved in most Dothideomycetes and upregulated during infection in L. maculans, suggesting a possible function in response to oxidative stress.

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 3ADON Population of Fusarium graminearum Found in North Dakota Is More Aggressive and Produces a Higher Level of DON than the Prevalent 15ADON Population in Spring Wheat

Fusarium head blight (FHB) is primarily caused by Fusarium graminearum in North America. Isolates of F. graminearum can be identified as one of three chemotypes: 3-acetyl-deoxynivalenol (3ADON), 15-acetyl-deoxynivalenol (15ADON), and nivalenol (NIV). In this study, we characterized F. graminearum isolates collected in 1980 to 2000 (old collection) and in 2008 (new collection) from North Dakota and found a 15-fold increase of 3ADON isolates in the new collection. Evaluation of randomly selected 3ADON isolates and 15ADON isolates on three spring wheat genotypes (Grandin, Steele-ND, and ND 2710) by single-floret inoculation indicated that the 3ADON population caused a higher disease severity and produced more DON at a significant level than the 15ADON population on Grandin (susceptible to FHB) and ND 2710 (with FHB resistance from Sumai 3). However, no significant differences in disease severity and DON production were observed between the two populations on Steele-ND (with moderate resistance from Triticum dicoccoides). The 3ADON isolates also exhibited a higher DON production in rice culture and produced more spores on agar media than the 15ADON isolates, suggesting a fitness advantage of the newly emerging 3ADON population over the prevalent 15ADON population. Population genetic analyses using DNA markers revealed a significant genetic differentiation between the two populations. The information obtained in this study could have an impact on development of FHB-resistant wheat cultivars and disease management.

A molecular genetic map and electrophoretic karyotype of the plant pathogenic fungus Cochliobolus sativus

A molecular genetic map was constructed and an electrophoretic karyotype was resolved for Cochliobolus sativus, the causal agent of spot blotch of barley and wheat. The genetic map consists of 27 linkage groups with 97 amplified fragment length polymorphism (AFLP) markers, 31 restriction fragment length polymorphism (RFLP) markers, two polymerase chain reaction amplified markers, the mating type locus (CsMAT), and a gene (VHv1) conditioning high virulence on barley cv. Bowman. These linkage groups covered a map distance of 849 cM. The virulence gene VHv1 cosegregated with six AFLP markers and was mapped on one of the major linkage groups. Fifteen chromosome-sized DNAs were resolved in C. sativus isolates ND93-1 and ND9OPr with contour-clamped homogeneous electric field (CHEF) electrophoresis combined with telomere probe analysis of comigrating chromosome-sized DNAs. The chromosome sizes ranged from 1.25 to 3.80 Mbp, and the genome size of the fungus was estimated to be approximately 33 Mbp. By hybridizing genetically mapped RFLP and AFLP markers to CHEF blots, 25 of the 27 linkage groups were assigned to specific chromosomes. The barley-specific virulence locus VHv1 was localized on a chromosome of 2.80 Mbp from isolate ND9OPr in the CHEF gel. The total map length of the fungus was estimated to be at least 1,329 cM based on the map distance covered by the linked markers and the estimated gaps. Therefore, the physical to genetic distance ratio is approximately 25 kb/cM. Construction of a high-resolution map around target loci will facilitate the cloning of the genes conferring virulence and other characters in C. sativus by a map-based cloning strategy.

Development and characterization of expressed sequence tag-derived microsatellite markers for the wheat stem rust fungus puccinia graminis f. sp. tritici

Puccinia graminis f. sp. tritici is the causal agent of stem rust disease in wheat. The rust fungus has caused devastating disease epidemics throughout history and is still posing a potential threat to wheat production in some regions of the world due to the appearance of new races. To develop microsatellite or simple sequence repeat (SSR) markers for use in population genetics studies, a total of 60,579 expressed sequence tag (EST) sequences (reads) generated from P. graminis f. sp. tritici were screened for tandemly repeated di- and tri-nucleotide units using a bioinformatics approach and 708 unisequences containing putative SSR loci with six or more repeat units were identified. Flanking primers were designed for 384 unique SSR loci, which mapped to different locations of the draft genome sequence of the fungus. Of the 384 primer pairs tested, 72 EST-SSR markers were eventually developed, which showed polymorphism among 19 isolates of P. graminis f. sp. tritici and 4 isolates of P. graminis f. sp. secalis evaluated. Thirty-two of the SSR loci were also evaluated in three other rust fungi (P. triticina, P. hordei, and P. coronata f. sp. hordei) for cross-species transferability. These SSR markers derived from ESTs will be useful for characterization of population structures and for gene mapping in P. graminis.

RNA‐mediated gene silencing in the cereal fungal pathogen Cochliobolus sativus

A high-throughput RNA-mediated gene silencing system was developed for Cochliobolus sativus (anamorph: Bipolaris sorokiniana), the causal agent of spot blotch, common root rot and black point in barley and wheat. The green fluorescent protein gene (GFP) and the proteinaceous host-selective toxin gene (ToxA) were first introduced into C. sativus via the polyethylene glycol (PEG)-mediated transformation method. Transformants with a high level of expression of GFP or ToxA were generated. A silencing vector (pSGate1) based on the Gateway cloning system was developed and used to construct RNA interference (RNAi) vectors. Silencing of GFP and ToxA in the transformants was demonstrated by transformation with the RNAi construct expressing hairpin RNA (hpRNA) of the target gene. The polyketide synthase gene (CsPKS1), involved in melanin biosynthesis pathways in C. sativus, was also targeted by transformation with the RNAi vector (pSGate1-CsPKS1) encoding hpRNA of the CsPKS1 gene. The transformants with pSGate1-CsPKS1 exhibited an albino phenotype or reduced melanization, suggesting effective silencing of the endogenous CsPKS1 in C. sativus. Sectors exhibiting the wild-type phenotype of the fungus appeared in some of the CsPKS1-silenced transformants after subcultures as a result of inactivation or deletions of the RNAi transgene. The gene silencing system established provides a useful tool for functional genomics studies in C. sativus and other filamentous fungi.

Characterization of Puccinia psidii isolates in Hawaii using microsatellite DNA markers

We studied genetic variation in Puccinia psidii, a newly introduced rust fungus in Hawaii, using microsatellite markers. All 36 Hawaiian P. psidii isolates analyzed had the same genotype at 11 microsatellite loci, while three genotypes were identified among four isolates from Florida and 11 genotypes among 18 isolates from Brazil. One of the isolates from Florida had 95% similarity to the Hawaiian isolates. We conclude that the genetically homogeneous P. psidii isolates in Hawaii might have derived from the same strain originally introduced into the Islands and are closely related to one of the strains found in Florida.

Identification and characterization of DNA markers associated with a locus conferring virulence on barley in the plant pathogenic fungus Cochliobolus sativus

Abstract.Cochliobolus sativus is a plant pathogenic fungus that causes spot blotch on barley and wheat. Virulence of a pathotype-2 isolate (ND90Pr) on barley cultivar Bowman was previously determined to be controlled by a single locus. To identify DNA markers associated with this virulence locus, amplified fragment length polymorphism (AFLP) analysis was conducted on 104 progeny isolates derived from a cross between isolates ND90Pr (exhibiting high virulence on Bowman) and ND93-1 (exhibiting low virulence on Bowman). Among 115 AFLP markers identified, 14 were linked to the virulence locus VHv1 in isolate ND90Pr, six of which co-segregated with VHv1. Two (E-AG/M-CA-207 and E-AG/M-CG-121) of the six co-segregating AFLP markers were cloned and used to probe genomic DNAs from the fungal parents and progeny. Both markers hybridized only with DNAs from ND90Pr and the virulent progeny. These two cloned markers were also used as probes to survey field isolates of C. sativus collected from different regions of the world and again only hybridized to DNAs from isolates that had the same virulence phenotype as ND90Pr. The results of this study indicate that E-AG/M-CA-207 and E-AG/M-CG-121 are closely linked to VHv1 and are unique to isolates carrying the virulence locus. Development of a linkage group, coupled with the identification of closely linked molecular markers, will facilitate the cloning of the virulence gene VHv1 in C. sativus by map-based cloning.