2009 Annual Report
1a.Objectives (from AD-416)
One of the objectives of this research is to identify genes involved in asexual sporulation of wheat-biotype Phaeosphaeria nodorum. The genes that are influenced by near ultra-violet light in their expression and highly expressed and critical for asexual sporulation will be studied. The other objective is to identify the quantitative trait loci (QTL) associated with fungal aggressiveness in wheat-biotype Phaeosphaeria nodorum. The long-term objective of this project is to develop an improved understanding of how specific gene expressions affect asexual sporulation and aggressiveness in Phaeosphaeria nodorum. The knowledge can be applied to develop strategies for Stagonospora nodorum blotch disease (P. nodorum is the sexual stage of S. nodorum) control in wheat and other cereals.
1b.Approach (from AD-416)
Vegetative and sporulative cultures that have spontaneously segregated from a single wheat-biotype P. nodorum colony are isolated. The subtraction technique is used to enrich the differentially expressed gene sequences present in poly (A) RNAs of sporulative culture. The expression of specific genes in vegetative and sporulative samples is studied by Northern hybridization. Genetic transformation of vegetative culture is planned to study the gene(s) involved in asexual conidiation. In order to define the gene(s) responsible for sporulation in wheat-biotype P. nodorum, particular gene(s) are also silenced by the cloning of open reading frame (ORF) of the gene. The genes encoding the transcription factors responsible for asexual sporulation in wheat-biotype P. nodorum can be identified by using a knock-out procedure. All transcription factors gene sequences which can be identified in wheat-biotype P. nodorum will be used for gene knockouts to determine their roles in asexual sporulation in P. nodorum.
The quantitative trait loci (QTL) associated with pathogen aggressiveness in wheat-biotype P. nodorum will be studied by a sexual crossing between two isolates with different aggressiveness levels and analyzing the segregation of molecular markers, functional genes and pathogen aggressiveness in their progeny. Polymorphisms in two parental isolates are detected with RAPD (Figure 1), RFLP, SSR, EST and AFLP techniques. The genetic linkage maps are analyzed and constructed with Joinmap 3.0 version software (www.biometris.nl). The segregation data of pathogen aggressiveness in the population are analyzed and the QTL mapping is produced by using an interval mapping method (MapQTL 5 software, www.biometris.nl).
Analyzed the segregation of 10 functional genes in the progeny of a P. nodorum sexual crossing. Specific primers were designed based on the sequence diversity and conditions for amplification of specific gene fragments explored. The linkage of these gene loci with other arbitrary markers were determined to complete the genetic map.
Identified light response genes and their expressions in wheat-biotype P. nodorum. There were eight hypothetic genes (SNOG series including 02675, 04238, 08907, 09297, 12044, 12501, 14195, and 15921), which encoded polypeptides with conserved light responsive domain, in P. nodorum. The complete nucleotide sequences of these eight genes were identified by designing specific primers, amplifying, and sequencing analysis. The messages represented these genes were extracted, amplified, and sequenced. It appeared that all these eight genes were expressed well in either sporulated or non-sporulated P. nodorum cultures grown under different light and dark conditions.
The structure of the light responsive white collar-1 (wc-1) gene (SNOG_12044) was identified. The polypeptide had three conserved light responsive domains. Multiple expressions of the wc-1 gene in wheat-biotype P. nodorum were discovered. The wc-1 gene would produce 6 different sizes of proteins, which might activate different sets of light dependent genes for pigment synthesis and sporulaton, and maintain day-night circadian clocks in fungi. This multiple expressions from a single gene phenomenon was common in vertebrates including human and viruses genes, but rare in fungi. It is the first time such a gene found in P. nodorum.
The structure of the light responsive white collar-2 (wc-2) gene (SNOG_14195) was identified. The polypeptide encoded by this gene had one conserved light responsive domain and would maintain a complex structure with wc-1 polypeptide for proper function. The gene and its product were well conserved in cereal Phaeosphaeria pathogens. However, some Phaeosphaeria pathogens derived from wild barley were highly diverted genetically.
Expression of light responsive genes in wheat-biotype P. nodurm. Spore production and dissipation are important factors for plant disease epidemic. Expression of light responsive genes, wc-1 and wc-2, are reported to be responsible for sporulation, pigmentation and day/night circadian cycle in fungi. The structures of the wc-1 and wc-2 genes in P. nodorum were isolated and studied. It appeared that a single wc-1 gene could produce six different sizes of proteins in Phaeosphaeria species. The difference in protein structure might provide specific regulations of other gene expressions under different environments, which led to different characters. The findings will be of interest to scientists in academic area.
Construction of a genetic linkage map of wheat-biotype Phaeosphaeria nodorum. Gene linkage maps are useful for identifying genetic locations of phenotypic traits in an organism and understanding the physiological and biochemical mechanism. Segregation of more than 300 arbitrary markers and 10 functioning genes were used to construct the genetic map of P. nodorum. The results of biological and pathological studies in the future would help the identification of genes responsible for asexual sporulation and pathogen aggressiveness in wheat-biotype P. nodorum.
Malkus, A., Song, Q., Cregan, P.B., Arseniuk, E., Ueng, P.P. 2009. Genetic linkage map of Phaeosphaeria nodorum, the causal agent of stagonospora nodorum blotch disease of wheat. European Journal of Plant Pathology. 124:681-690.
Wang, C., Chang, P., Lin, Y., Malkus, A., Ueng, P.P. 2009. Group I introns in small subunit ribosomal DNA of several Phaeosphaeria species. Botanical Review. 50:137-147.