Molecular approaches to analyze horizontal resistance among plant species (maize, soybean, and Arabidopsis) to syntenic fusarial pathogens (F. graminearum and F. virguliforme).

摘要

Numerous evidences showed that the resistance to syntenic fungal species Fusarium graminearum and Fusarium virguliforme infection in plants was partial, quantitative, and strongly influenced by environmental conditions. However, the molecular mechanisms underlying the resistance are still not fully understood. The objectives of this dissertation were to investigate the molecular mechanisms underlying the resistance in different plant species (mainly in maize, soybean, and Arabidopsis); identify and clone disease resistance genes; decipher their quantitative responses to Fusarial pathogens and mycotoxin deoxynivalenol (DON); identified genes that were transcriptionally regulated when the plants were treated with the pathogens and toxin. Methods were used to achieve these objectives including mapping, isolation, characterization, and functional analyses of genes implicated in disease resistance; analysis of microsynteny at their encoding loci; and identification of orthologs that might be associated with resistance to pathogen Fusaria across plant species etc. First, a maize gene that encoded a putative guanylyl cyclase-like protein (ZmGC1; EC 4.6.1.2) was characterized and shown to be associated with resistance to Gibberella ear rot caused by F. graminearum. The putative ZmGC1 amino acid sequence was 53% identical and 65% similar to AtGC1, one of the two Arabidopsis proteins known to possess an active and substrate specific guanylyl cyclase enzymatic function. Using a probe derived from the maize guanylyl cyclase-like gene (Zmgc1) to screen a recombinant inbred population developed from 'CO387' (a partially resistant variety) X 'CG62' (a partially susceptible variety), several polymorphic molecular markers were identified and four of them were significantly associated with Gibberella ear rot resistance in different environments. Polymorphisms were functional since the amount of Zmgc1 transcripts accumulating in ears increased more quickly and to a higher amount in the resistant genotype compared to the susceptible genotype after inoculation with F. graminearum . Furthermore, transcripts were responding to fungicidal activity when the maize seedlings (CO387) were treated with a fungicide, probenazole (3-allyloxy-1,2-benzothiazole-1,1-dioxide, PBZ). The transcript abundance (TA) of the maize Zmgc1 gene was increased more than 10 fold 8 hours after PBZ treatment. In contrast, the TA of Zmnbslrr1 (a NBS-LRR gene) transcript was significantly reduced in the Gibberella ear rot resistant genotype CO387 after the treatment with PBZ in the time-course study. Therefore, natural resistance and fungicide induced resistance may share common transcript abundance changes in maize plants.;Second, to examine global effects of Fusarial pathogens on transcript abundance, cDNA microarrays from the model plant Arabidopsis thaliana were used. After Arabidopsis thaliana cv 'Columbia' was infested with Fusarium virguliforme, 168 transcripts were increased, nearly four times more than that of decreased. A. thaliana seedling growth was reduced by the pathogen in a proportional response to increasing spore concentrations. A set of putative resistance pathways involved in responding to the pathogen infection in A. thaliana was identified. Functional analyses of the orthologs of the responding genes between soybean and A. thaliana showed that the resistance responses had both common elements in some pathways (primary metabolism) and species specific differences in others (secondary metabolism). For example, the phenylpropanoid pathway response was different between the two species. In contrast to soybean, the phenylpropanoid pathway was not fully activated during the resistance response in Arabidopsis. Therefore, soybean and Arabidopsis did not share completely overlapping strategies in the specific pathways induced during resistance to F. virguliforme.;Resistance to Fusarial toxins is a common mechanism for plant resistance. The mycotoxin deoxynivalenol (DON), produced by Gibberella zeae (the teleomorph of F. graminearum), was known to be both a virulence factor in the pathogenesis of wheat and an inhibitor of Arabidopsis seed germination. A. thaliana seedling growth was reduced by the toxin in a proportional response to increasing concentrations. A parallel comparison with a set of resistance pathways involved in response to the DON toxicity in A. thaliana was performed. The alterations of transcript abundances in the Arabidopsis plants treated with the toxin suggest that DON plays a significant role affecting the key primary metabolisms in Arabidopsis plants. The alterations ranged from the protein metabolism to redox production. New putative resistance pathways involved in responding to both pathogen and DON infestation in soybean and A. thaliana were described.