Research Accomplishment Reports 2007

Genomic Studies of the Model Phytopathogenic Fungus Magnaporthe grisea

M. Farman
Department of Plant Pathology

 

Project Description

Previous work on the molecular basis for telomere hypervariability in magnaporthe oryzae isolates that infect perennial ryegrass. has shown that the chromosome ends of these isolates are organized quite differently to those in isolates from rice. Specifically, the subtelomere regions contain tandem arrays of three types of retrotransposon-like sequences. These sequences are present only at the chromosome ends and, therefore, we named them magnaporthe oryzae telomere exclusive repeats, or moters. Moter1 is 4.6 Kb in length and codes for a reverse transcriptase, moter2 is 1.7 Kb and codes for a hypothetical protein of 204 amino acids, and moter3 is 4.95 Kb long and appears to be a recombinant of moter1 and moter2. We cloned several de novo telomeres that arose during culture of LPKY97-1, a ryegrass pathogen, leading to identification of two newly transposed moter1 elements. Both new elements had inserted into the existing telomere array at positions that were within three repeat units of the chromosome-unique sequences. This provides convincing evidence that moter1 is an active transposon that inserts specifically at chromosome ends.

In 2007, we focused on: 1) determining the nature of genomic rearrangements leading to changes in the sizes of terminal restriction fragments; and 2) testing whether the presence of moter elements at the chromosome ends affects telomere integrity. We created 200 single-spore isolates of magnaporthe strain lpky97-1, and analyzed telomere profiles for 100. Nearly all of the single-spore isolates showed changes in their telomere restriction profile relative to the parent strain. By cloning telomeres from these strains, we found that many of the telomere rearrangements in single spore isolates are not associated with moter transposition. Instead, they often appeared to arise via terminal truncations. In at least one case, the truncated chromosome end invaded an internal chromosome region and captured a sequence, leading to its duplication at the telomere. We are currently cloning some of these novel ends to determine if they arose from ends formerly containing moter elements. We are using the single-spore isolate collection and clones of chromosome-unique (cu) sequences flanking moter elements to study the inherent stability of individual chromosome ends. We have examined three chromosome ends. We produced strains containing either moter1, moter2 or both elements to examine the interdependence of the two elements for transposition activity. We backcrossed moter-containing isolates to a magnaporthe strain lacking these elements. We have isolated over 60 progeny from a new cross between the moter-containing strain, fh and the moter-less strain, 2539. Progeny strains have been genotyped for mating-type to allow selection of sexually-compatible strains to backcross with 2539. Telomere-enriched clone libraries were constructed for strain 2539 and progeny isolates 3-1 and 4-10. One telomere has been cloned from each of 2539 and 3-1. To determine the molecular basis for strain-to-strain differences in the sizes of the major moter-hybridizing bands, moter-moter junctions were amplified from several magnaporthe strains.

Impact

In most eukaryotic microbes, the chromosome ends contain genes that are important for niche adaptation. This is especially evident in pathogens, because their subtelomere regions frequently contain genes that tend to trigger host defenses. This association is true also for M. oryzae, a fungus that causes devastating diseases of rice, other crops and turfgrasses. In M. oryzae, the ability to infect particular host plants is controlled by avirulence (AVR) genes. Almost 50% of the known AVR genes in this fungus map very near to the chromosome ends. It follows that telomere instability could have a profound impact on the pathogenic capabilities of M. oryzae. Therefore, understanding mechanisms of telomere instability is expected to yield new insights into the mechanisms by which this fungus evades host recognition to defeat resistant rice/perennial rygrass varieties. This information will be vitally important for developing sound strategies for the management and, hopefully, the eventual eradication of diseases caused by this fungus.

Publications

Farman M., 2007, Telomeres in the rice blast fungus Magnaporthe oryzae: the world of the end as we know it. FEMS Lett. 273: 125-132.

Vincelli P., and Farman, M., 2007, Update on fungicide resistance in gray leaf spot. Golf Course Management, February, 124-127.

Clutterbuck, J. and Farman, M., 2007, Aspergillus nidulans linkage map and genome sequence: closing some gaps and adding telomeres. In The Aspergilli: genomics, medical aspects, biotechnology and research methods. edited by G. Goldman and S. Osmani. CRC Press-Taylor and Francis Group, LLC, Boca Raton, FL.

Chakrabarty R., Banerjee R., Chung, S-M., Farman M. L., Citovsky V., Hogenhout, S. A. Tzfira T., Goodin M. M., 2007, pSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: probing Nicotiana benthamiana-virus interactions. Molec. Plant Microbe Interact. 20:740-750.

Meng Y, Patel G, Heist M, Betts M, Tucker, Galadima N, Donofrio N, Brown D, Mitchell T, Li L, Xu J R, Orbach M, Thon M, Dean R, Farman M, 2007, A systematic analysis of T DNA insertion events in Magnaporthe oryzae. Fungal Genet. Biol. 44:1050-1064.

Betts M, Tucker S, Galadima N, Meng Y, Patel G, Li L, Donofrio N, Floyd A, Nolin S, Brown D, Mandel A, Mitchell T, Xu J-R, Dean R, Farman M, Orbach M, 2007, Development of a high throughput transformation system for insertional mutagenesis in Magnaporthe oryzae. Fungal Genet. Biol. 44:1035-1049.