Identification and Genesis of a Cluster of Fungal Pathogenicity Genes

这项研究有可能在真菌中发现一类新的致病基因，并进一步了解这些基因的基因组组织和传递。尽管以细菌致病岛为代表的基因群的水平转移被认为是动植物细菌病原体中的一种现象和进化动力，但它在真菌致病能力进化中的作用却存在争议。PEP簇是一组与豌豆红球线虫致病相关的基因，对其起源的研究将有助于理解豌豆致病基因的进化，有助于确定真菌能否通过水平转移获得致病基因。PEP簇位于一条1.6Mb的条件可支配(CD)染色体上，这对培养物的生长是必不可少的，但对于特定生境的生长是必需的，在这种情况下是豌豆组织。PEP原型簇包含6个在豌豆侵染过程中表达的基因。对25个田间分离物的调查表明，所有高毒力分离物都具有该簇的所有成员，而非强毒分离物中没有一个包含整个簇。最近，在原型PEP簇两侧的区域发现了另外六个在感染过程中表达的ORF。用聚类算法比较这些基因和21个非Cd(N)染色体基因的密码子偏好性时，发现了与基因来源高度相关的两组不同的基因。PEP区基因和其他染色体基因在DNA结构上的这些差异与PEP簇，甚至整个CD染色体是外源起源的假设是一致的。本项目的目的是进一步确定PEP簇中的每个基因对毒力的贡献，包括在簇两侧新发现的ORF，并确定这些基因及其所在的CD染色体是否是通过水平转移获得的。为了解决水平基因转移的问题，将获得来自N和CD染色体的大约100个额外基因的DNA序列，并将利用多变量分析比较它们的特征(例如G+C含量、密码子使用、二核苷酸和三核苷酸的频率以及3：1二核苷酸偏向)。将确定具有不同致病表型的分离物中PEP同源物的物理位置和组织，以深入了解可能导致簇形成和/或碎裂的事件。最后，我们将分析来自CD染色体的PEP基因和其他基因的系统发育分布，以扩展初步结果，该结果显示了不连续的分布，这一结果与水平基因转移一致。这个项目解决了真菌基因组进化的基本问题。此外，对致病性状的鉴定和关于真菌如何获得致病性状的知识可能会直接影响目前控制这些农业害虫的方法，并有助于设计新的预防植物病害的策略。来自亚利桑那大学和路德学院的本科生将参与这项研究。路德学院是参与植物病理学系“冬季实习计划”的六所文科院校之一。这笔赠款的资金将使这些路德学院的学生能够在他们的家乡机构利用当代技术进行实践研究。

This research has the potential to identify a new class of pathogenicity genes in fungi and to further the understanding of the genomic organization and transmission of these genes. Whereas the horizontal transfer of groups of genes, as exemplified by bacterial pathogenicity islands, is well accepted as a phenomenon and an evolutionary driving force in bacterial pathogens of plants and animals, its role in the evolution of fungal pathogenicity is controversial. The studies on the genesis of the PEP cluster, a group of genes involved in the pathogenicity of the fungus Nectria haematococca on pea, will contribute to the understanding of the evolution of pathogenicity and help to determine whether fungi can acquire pathogenicity genes by horizontal transfer. The PEP cluster is located on a 1.6-Mb conditionally dispensable (CD) chromosome, which is dispensable for growth in culture but is required for growth in specific habitats, in this case pea tissue. The prototype PEP cluster contains six genes that are expressed during the infection of pea. A survey of 25 field isolates has revealed that all highly virulent isolates have all members of the cluster, while none of the non-virulent isolates contains the entire cluster. Recently, six additional ORFs, which are expressed during infection, have been identified in the regions flanking the prototype PEP cluster. When the codon bias of these genes and 21 genes from non-CD (N) chromosomes were compared using a cluster algorithm, two distinct groups with a high degree of correlation to the sources of the genes were identified. These differences in DNA structure between the genes in the PEP region and the genes from the other chromosomes are consistent with the hypothesis that the PEP cluster, and perhaps the entire CD chromosome, are of exogenous origin. The objectives of this project are to characterize further the contributions made to virulence by each gene in the PEP cluster, including the newly identified ORFs in the regions flanking the cluster, and to determine if these genes and the CD chromosome upon which they reside were acquired by horizontal transfer. To address the question of horizontal gene transfer, the DNA sequences of approximately one hundred additional genes from both the N and CD chromosomes will be obtained and their characteristics (e. g. G+C content, codon usage, frequency of di- and tri- nucleotides, and 3:1 dinucleotide bias) will be compared utilizing multivariate analysis. The physical locations and organization of the PEP homologues in isolates with different pathogenicity phenotypes will be determined to gain insight into events that may have lead to the formation and/or fragmentation of the cluster. Finally, the phylogenetic distribution of the PEP genes and other genes from the CD chromosome will be analyzed to extend preliminary results that showed a discontinuous distribution, a result that is consistent with horizontal gene transfer. This project addresses basic questions on the evolution of fungal genomes. In addition, the identification of pathogenicity traits and knowledge about how fungi acquire them may directly affect the current methods for the control of these agricultural pests and aid in the design of new strategies to prevent plant diseases. Undergraduates from the University of Arizona and from Luther College, one of six liberal arts colleges participating in the Department of Plant Pathology's "Winter Internship Program", will participate in this research. Funds from this grant will enable these Luther College students to carry out hands-on research utilizing contemporary technologies at their home institution.