Peter P. Ueng

The CTB1 Gene Encoding a Fungal Polyketide Synthase Is Required for Cercosporin Biosynthesis and Fungal Virulence of Cercospora nicotianae

Cercosporin is a light-activated, non-host-selective toxin produced by many Cercospora fungal species. In this study, a polyketide synthase gene (CTB1) was functionally identified and molecularly characterized to play a key role in cercosporin biosynthesis by Cercospora nicotianae. We also provide conclusive evidence to confirm the crucial role of cercosporin in fungal pathogenesis. CTB1 encoded a polypeptide with a deduced length of 2,196 amino acids containing a keto synthase (KS), an acyltransferase (AT), a thioesterase/claisen cyclase (TE/CYC), and two acyl carrier protein (ACP) domains, and had high levels of similarity to many fungal type I polyketide synthases. Expression of a 6.8-kb CTB1 transcript was highly regulated by light and medium composition, consistent with the conditions required for cercosporin biosynthesis in cultures. Targeted disruption of CTB1 resulted in the loss of both CTB1 transcript and cercosporin biosynthesis in C. nicotianae. The ctb1-null mutants incited fewer necrotic lesions on inoculated tobacco leaves compared with the wild type. Complementation of ctb1-null mutants with a full-length CTB1 clone restored wild-type levels of cercosporin production as well as the ability to induce lesions on tobacco. Thus, we have demonstrated conclusively that cercosporin is synthesized via a polyketide pathway, and cercosporin is an important virulence factor in C. nicotianae. The results also suggest that strategies that avoid the toxicity of cercosporin will be useful in reduction of disease incidence caused by Cercospora spp.

Foci of stagonospora nodorum blotch in winter wheat before canopy development.

ABSTRACT Stagonospora nodorum blotch (SNB) often develops explosively on upper leaves and glumes of wheat. Inoculum for late season infections may arise from early disease foci in the lower canopy or from recent immigration of wind-dispersed ascospores. Research was conducted to determine if foci of SNB are present and secondary spread has occurred in fields before tiller elongation. We determined the incidence of infection by Stagonospora nodorum for plants sampled at the mid-tillering stage in 96 1-m(2) quadrats in each of two fields. Isolates of S. nodorum were recovered from 32 quadrats, one per infected plant where possible. Multilocus restriction fragment length polymorphism haplotypes were determined for each isolate. Of 55 isolates collected from one field, there were 22 distinct haplotypes. Diseased plants were aggregated in both fields; aggregates sometimes extended to adjacent quadrats. Plants within aggregates were often infected by the same haplotype, suggesting that secondary spread had occurred. Foci overlapped because some aggregates were infected by more than one haplotype. Our results show that genetically diverse populations of S. nodorum were already established in fields before canopy development and were comprised of sometimes overlapping foci undergoing clonal expansion.

Serological and Molecular Characterization of a High Temperature-recovered Virus Belonging to Tospovirus Serogroup IV

A serologically and cytologically distinct gloxinia tospovirus (HT-1) previously isolated from a gloxinia plant infected with Impatiens necrotic spot virus (INSV) when propagated in a high-temperature environment was characterized. Rabbit antisera produced for INSV and Tomato spotted wilt virus (TSWV) nucleocapsids (N) failed to react with HT-1 proteins in western blot analysis. The HT-1 antibodies reacted strongly with homologous antigen but failed to react with INSV and TSWV. However, the HT-1 antiserum reacted in ELISA with Watermelon silver mottle virus (WSMV) from Taiwan and in western blot analysis with the WSMV N protein. A reciprocal test showed that the antiserum prepared against the N protein of WSMV also reacted with the HT-1 N protein in both ELISA and western blot analysis. DNA probes derived from the N gene of HT-1 or WSMV hybridized to RNAs prepared from plants infected with either virus. Stronger signals were obtained with homologous than with heterologous reactions. Neither probe detected INSV or TSWV. The M and S RNAs of HT-1 were sequenced. The M RNA contains two open reading frames (ORF) ; one in the sense orientation encoding a nonstructural (NSm) protein of 308-amino-acids (aa) and the other in the ambisense orientation, a 1122-aa precursor of Gl and G2 glycoproteins. The S RNA also contains two ORFs ; one in the sense orientation encoding a nonstructural (NSs) protein of 439 aa and the other in the ambisense orientation, an N protein of 277 aa. HT-1 is distantly related to INSV and TSWV as shown by low nucleotide (40–52%) and amino acid (28–48%) similarities in the four ORF sequences. The HT-1 virus shares high nucleotide (76–81%) and amino acid (85–92%) similarities with WSMV and peanut bud necrosis virus (PBNV). Based on the serological properties and sequence data, we propose that HT-1 is a distinct species of serogroup IV in the genus Tospovirus. This is the first time that a tospovirus similar to those found in the Far East and in Southeast Asia has been identified in the US.

Resistance to Cucumber mosaic virus in Gladiolus plants transformed with either a defective replicase or coat protein subgroup II gene from Cucumber mosaic virus.

Transgenic Gladiolus plants that contain either Cucumber mosaic virus (CMV) subgroup I coat protein, CMV subgroup II coat protein, CMV replicase, a combination of the CMV subgroups I and II coat proteins, or a combination of the CMV subgroup II coat protein and replicase genes were developed. These plants were multiplied in vitro and challenged with purified CMV isolated from Gladiolus using a hand-held gene gun. Three out of 19 independently transformed plants expressing the replicase gene under control of the duplicated CaMV 35S promoter were found to be resistant to CMV subgroup I. Three out of 21 independently transformed plants with the CMV subgroup II coat protein gene under control of the Arabidopsis UBQ3 promoter were resistant to CMV subgroup II. Eighteen independently transformed plants with either the CMV subgroup I coat protein or a combination of CMV subgroups I and II coat proteins were challenged and found to be susceptible to both CMV subgroups I or II. Virus resistant plants with the CMV replicase transgene expressed much lower RNA levels than resistant plants expressing the CMV subgroup II coat protein. This work will facilitate the evaluation of virus resistance in transgenic Gladiolus plants to yield improved floral quality and productivity.

QTL controlling partial resistance to Stagonospora nodorum leaf blotch in winter wheat cultivar Alba

Stagonospora nodorum blotch (SNB) is an important foliar and glume disease in cereals. Inheritance of SNB resistance in wheat appears quantitative. The development of partially resistant cultivars seems to be the only effective way to combat the pathogen. Partial resistance components like length of incubation period (INC), disease severity (DIS) and length of latent period (LAT) were evaluated on a population of doubled-haploids derived from a cross between the partially resistant cultivar Alba and the susceptible cultivar Begra. Experiments were conducted in controlled environments and the fifth leaf was examined. Molecular analyses were based on bulked segregant analyses (BSA) and screening with 240 microsatellites DNA markers. The QTL analysis revealed QTL on chromosome 6AL (designated as QSnl.ihar-6A) and putative QTL on chromosome 6D. The QSnl.ihar-6A accounted for 36% of the phenotypic variance for DIS and 14% for INC. The putative QTL accounted for 10% of the variability in INC and 8% of DIS components of SNB resistance.

Sequence diversity of mating-type genes in Phaeosphaeria avenaria

Abstract. Phaeosphaeria avenaria, one of the causal agents of stagonospora leaf blotch diseases in cereals, is composed of two subspecies, P. avenaria f. sp. triticea (Pat) and P. avenaria f. sp. avenaria (Paa). The Pat subspecies was grouped into Pat1–Pat3, based on restriction fragment length polymorphism (RFLP) and ribosomal DNA (rDNA) internal transcribed spacer (ITS) sequences in previous studies. Mating-type genes and their potential use in phylogeny and molecular classification were studied by DNA hybridization and PCR amplification. The majority of Pat1 isolates reported to be homothallic and producing sexual reproduction structures on cultural media had only the MAT1-1 gene. Minor sequence variations were found in the conserved region of MAT1-1 gene in Pat1 isolates. However, both mating-type genes, MAT1-1 and MAT1-2, were identified in P. avenaria isolates represented by ATCC12277 from oats (Paa) and the Pat2 isolates from foxtail barley (Hordeum jubatum L.). Cluster analyses based on mating-type gene conserved regions revealed that cereal Phaeosphaeria is not phylogenetically closely related to other ascomycetes, including Mycosphaerella graminicola (anamorph Septoria tritici). The sequence diversity of mating-type genes in Pat and Paa supports our previous phylogenetic relationship and molecular classification based on RFLP fingerprinting and rDNA ITS sequences.