Rubella S. Goswami

Targeted gene replacement in fungi using a split-marker approach.

Targeted gene replacement is one of the primary strategies for functional characterization of fungal genes and several methods have been developed for this purpose over the years. The increased availability of genome sequence information in the present times has enabled wider adoption of protocols based on the knowledge of the gene sequence and its surrounding region. Among such targeted gene replacement approaches, the spilt-marker method has gained popularity in filamentous fungi. This method involves only two rounds of PCR and does not require any subcloning. It is based on the availability of a marker gene (e.g., the hygromycin gene) and sequences of the gene of interest, as well as around 1 kb long regions flanking the gene on either side. The technique includes PCR amplification of the flanking regions of the gene of interest and the marker gene followed by a fusion PCR which leads to the creation of two molecular cassettes, each containing a part of the marker gene fused to one flanking region. These molecular cassettes are then simultaneously used for transformation of protoplasts. Three homologous recombination events, one within each flanking region and one in the marker gene, lead to the replacement of the gene of interest with a functional marker gene. The transformants are then grown on selective media and emerging colonies can be screened for presence of the marker and absence of the gene being replaced using various methods.

Characterization and Pathogenicity of Rhizoctonia solani Isolates Affecting Pisum sativum in North Dakota

Acreage of dry field pea (Pisum sativum) in North Dakota has increased approximately eightfold from the late 1990s to the late 2000s to over 200,000 ha annually. A coincidental increase in losses to root rots has also been observed. Root rot in dry field pea is commonly caused by a complex of pathogens which included Fusarium spp. and Rhizoctonia solani. R. solani isolates were obtained from roots sampled at the three- to five-node growth stage from North Dakota pea fields and from symptomatic samples received at the Plant Diagnostic Lab at North Dakota State University in 2008 and 2009. Using Bayesian inference and maximum likelihood analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA), 17 R. solani pea isolates were determined to belong to anastomosis group (AG)-4 homogenous group (HG)-II and two isolates to AG-5. Pathogenicity of select pea isolates was determined on field pea and two rotation hosts, soybean and dry bean. All isolates caused disease on all hosts; however, the median disease ratings were higher on green pea, dry bean, and soybean cultivars when inoculated with pea isolate AG-4 HG-II. Identification of R. solani AGs and subgroups on field pea and determination of relative pathogenicity on rotational hosts is important for effective resistance breeding and appropriate rotation strategies.

First Report of Ascochyta Blight Caused by QoI-Resistant Isolates of Ascochyta rabiei in Chickpea Fields of Nebraska and South Dakota

Ascochyta blight, caused by Ascochyta rabiei, is a serious disease of chickpea (Cicer arietinum) and fungicide applications are used to manage the disease in the North Central plains (4). During the 2010 growing season, a commercial field near Stanley, SD was treated with pyraclostrobin (Headline, BASF, NC) and called a management failure by the grower. Similarly, limited efficacy of pyraclostrobin was observed in an ascochyta research trial near Scotts Bluff, NE. In both locations, symptoms and signs consistent with A. rabiei infection existed on leaves, stems, and pods; namely, circular brown lesions with concentric rings of dark brown pycnidia. Symptomatic samples were collected, disinfected with 95% ethanol for 1 min, rinsed with sterile water, placed in 0.5% NaOCl for 1 min, and rinsed again with sterile water for 1 min (4). Samples were air dried, placed on potato dextrose agar (PDA) plates for 3 to 7 days, and colonies with morphological characteristics typical of A. rabiei were single-spored and transferred to new PDA plates and incubated for 7 to 14 days. Three and six putative A. rabiei isolates were obtained from South Dakota and Nebraska samples, respectively. Morphological characteristics were consistent with A. rabiei; cultures were brown with concentric rings of dark, pear-shaped pycnidia with an ostiole, and conidia were hyaline, single-celled, and oval-shaped (2). Comparison of the internal transcribed spacer (ITS) region amplified from the genomic DNA of 3-day-old liquid cultures using ITS4/ITS5 primers by BLASTN searches using the nr database in GenBank (Accession Number FJ032643) also confirmed isolates to be A. rabiei. Mismatch amplification mutation assay (MAMA) PCR was used for detection of sensitive and resistant isolates to QoI fungicides (1). Confirmation of the presence of the G143A mutation was carried out by cloning an mRNA fragment of the cytochrome b gene using cDNA synthesized from total RNA of A. rabiei and CBF1/CBR2 (1,3). Total RNA was extracted from 3-day-old liquid cultures and it was used instead of genomic DNA for this PCR to avoid large intronic regions commonly present in mitochondrial genes. The G143A mutation has previously been correlated with resistance to QoI fungicides in other fungal plant pathogens (3). Also, these isolates were determined to be QoI-resistant in vitro by PDA amended with a discriminatory dose of 1 μg/ml of azoxystrobin (4). To our knowledge, this is the first report of QoIresistant A. rabiei isolates causing infections on chickpeas in South Dakota and Nebraska. QoI-resistant isolates were reported in North Dakota and Montana in 2005 and 2007, respectively (4). Of nearly 300 isolates collected from these states from 2005 and 2007, approximately 65% were determined to be QoI resistant (4). The widespread occurrence of QoIresistant isolates and reduction of fungicide performance in fields led the North Dakota State University Cooperative Extension Service to actively discourage the use of QoI fungicides on chickpeas in North Dakota and Montana (4). It is likely that similar recommendations will need to be adopted in South Dakota and Nebraska for profitable chickpea production. References: (1) J. A. Delgado, 2012 Ph.D. Diss. Department of Plant Pathology, North Dakota State University. (2) R. M. Harveson et al. 2011. Online. Plant Health Progress doi:10.1094/PHP-2011-0103-01-DG. (3) Z. Ma et al. Pestic. Biochem. Physiol. 77:66, 2003. (4) K. A. Wise et al. Plant Dis. 93:528, 2009.

Colletotrichum lindemuthianum Races Prevalent on Dry Beans in North Dakota and Potential Sources of Resistance

Anthracnose caused by Colletotrichum lindemuthianum is one of the most important diseases of dry edible beans in the major production areas worldwide. This pathogen is highly variable, with numerous races. Disease management relies heavily on genetic resistance and use of clean seed. Genetic resistance is controlled by major resistance genes conferring protection against specific races of the pathogen. Therefore, knowledge of the pathogen population in a region is essential for effective screening of germplasm. Surveys were conducted for more than 6 years in North Dakota, the largest dry-bean-growing state in the United States, and seed samples submitted for certification were assessed to identify the C. lindemuthianum races prevalent in the region. A collection of commercial cultivars from different market classes of dry bean was also screened for resistance to these races. Disease incidence was found to be low in most years. However, in addition to the previously reported races of anthracnose 7, 73, and 89, two new races, 1153 and 1161, previously never reported in the United States, were identified and the commercial cvs. Montcalm, Avalanche, Vista, and Sedona where found to possess resistance to these races.

A resource for the in silico identification of fungal polyketide synthases from predicted fungal proteomes

The goal of this study was to develop a tool specifically designed to identify iterative polyketide synthases (iPKSs) from predicted fungal proteomes. A fungi-based PKS prediction model, specifically for fungal iPKSs, was developed using profile hidden Markov models (pHMMs) based on two essential iPKS domains, the β-ketoacyl synthase (KS) domain and acyltransferase (AT) domain, derived from fungal iPKSs. This fungi-based PKS prediction model was initially tested on the well-annotated proteome of Fusarium graminearum, identifying 15 iPKSs that matched previous predictions and gene disruption studies. These fungi-based pHMMs were subsequently applied to the predicted fungal proteomes of Alternaria brassicicola, Fusarium oxysporum f.sp. lycopersici, Verticillium albo-atrum and Verticillium dahliae. The iPKSs predicted were compared against those predicted by the currently available mixed-kingdom PKS models that include both bacterial and fungal sequences. These mixed-kingdom models have been proven previously by others to be better in predicting true iPKSs from non-iPKSs compared with other available models (e.g. Pfam and TIGRFAM). The fungi-based model was found to perform significantly better on fungal proteomes than the mixed-kingdom PKS model in accuracy, sensitivity, specificity and precision. In addition, the model was capable of predicting the reducing nature of fungal iPKSs by comparison of the bit scores obtained from two separate reducing and nonreducing pHMMs for each domain, which was confirmed by phylogenetic analysis of the KS domain. Biological confirmation of the predictions was obtained by polymerase chain reaction (PCR) amplification of the KS and AT domains of predicted iPKSs from V. dahliae using domain-specific primers and genomic DNA, followed by sequencing of the PCR products. It is expected that the fungi-based PKS model will prove to be a useful tool for the identification and annotation of fungal PKSs from predicted proteomes.

Gene cloning using degenerate primers and genome walking.

Gene cloning is the first step of targeted gene replacement for functional studies, discovery of gene alleles, and gene expression among other applications. In this chapter, we will describe a cloning technique suitable for fungal species where the genomic information and sequences available are limited. This strategy involves obtaining protein sequences of the gene of interest from various organisms to identify at least two conserved regions. Degenerate primers are designed from these two conserved regions and the resulting PCR products are sequenced. The sequence of the PCR products can be analyzed using suitable databases to determine their similarity to the gene/protein of interest. In cases where the entire gene cannot be cloned directly using these primers, this initial nucleotide sequence can be used as a template for further primer design and genome walking in both directions for either the cloning of a longer fragment or even the cloning of the complete gene. Here, we describe the partial cloning of a reducing polyketide synthase gene from the fungal plant pathogen Ascochyta rabiei using this strategy.