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MicroRNAs suppress NB domain genes in tomato that confer resistance to Fusarium oxysporum.

Ouyang S, Park G, Atamian HS, Han CS, Stajich JE, Kaloshian I, Borkovich KA - PLoS Pathog. (2014)

Bottom Line: One slmiR5300 target corresponds to tm-2, a susceptible allele of the Tomato Mosaic Virus resistance gene, supporting functions in immunity to a fungal pathogen.The observation that none of the targets correspond to I-2, the only known resistance (R) gene for F. oxysporum in tomato, supports roles for additional R genes in the immune response.Taken together, our findings suggest that Moneymaker is highly susceptible because its potential resistance is insufficiently expressed due to the action of miRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology and Microbiology, Institute for Integrative Genome Biology, University of California, Riverside, Riverside, California, United States of America.

ABSTRACT
MicroRNAs (miRNAs) suppress the transcriptional and post-transcriptional expression of genes in plants. Several miRNA families target genes encoding nucleotide-binding site-leucine-rich repeat (NB-LRR) plant innate immune receptors. The fungus Fusarium oxysporum f. sp. lycopersici causes vascular wilt disease in tomato. We explored a role for miRNAs in tomato defense against F. oxysporum using comparative miRNA profiling of susceptible (Moneymaker) and resistant (Motelle) tomato cultivars. slmiR482f and slmiR5300 were repressed during infection of Motelle with F. oxysporum. Two predicted mRNA targets each of slmiR482f and slmiR5300 exhibited increased expression in Motelle and the ability of these four targets to be regulated by the miRNAs was confirmed by co-expression in Nicotiana benthamiana. Silencing of the targets in the resistant Motelle cultivar revealed a role in fungal resistance for all four genes. All four targets encode proteins with full or partial nucleotide-binding (NB) domains. One slmiR5300 target corresponds to tm-2, a susceptible allele of the Tomato Mosaic Virus resistance gene, supporting functions in immunity to a fungal pathogen. The observation that none of the targets correspond to I-2, the only known resistance (R) gene for F. oxysporum in tomato, supports roles for additional R genes in the immune response. Taken together, our findings suggest that Moneymaker is highly susceptible because its potential resistance is insufficiently expressed due to the action of miRNAs.

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Co-expression of slmiRNAs and predicted targets in Nicotiana benthamiana leaves.A. Levels of target mRNAs. qRT-PCR was used to determine relative levels of predicted tomato mRNAs in N. benthamiana leaves expressing only empty vector; target mRNA and a control miRNA (slmiR166); target mRNA and the appropriate miRNA (slmiR482f or slmiR5300); or target mRNA and empty vector. Values were normalized to N. benthamiana actin. Errors are expressed as the standard error. *** indicates significant differences when compared to the corresponding control plants in the same treatments at p<0.001. B. Target protein levels. Total protein isolated from the samples in (A) was electrophoresed on SDS-PAGE gels and blotted onto nitrocellulose membranes. A FLAG antiserum was used to detect the tagged target proteins during western analysis as described in the Materials and Methods (top panels). A duplicate gel was Coomassie-stained and used as a loading control (bottom panels). Similar results were obtained for two biological replicates. C. Diagrammatic representation of target mRNA cleavage sites determined using 5′RACE. Thick black lines represent open reading frames (nucleotide numbering begins at the start codon indicated by the black arrow), and the putative miRNA interaction site is shown as a gray box, with the nucleotide position within the ORF indicated. The nucleotides shown in red in each mRNA target are mismatches with the corresponding miRNA. The number of sequenced 5′RACE clones corresponding to each site is indicated by vertical arrowheads.
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ppat-1004464-g005: Co-expression of slmiRNAs and predicted targets in Nicotiana benthamiana leaves.A. Levels of target mRNAs. qRT-PCR was used to determine relative levels of predicted tomato mRNAs in N. benthamiana leaves expressing only empty vector; target mRNA and a control miRNA (slmiR166); target mRNA and the appropriate miRNA (slmiR482f or slmiR5300); or target mRNA and empty vector. Values were normalized to N. benthamiana actin. Errors are expressed as the standard error. *** indicates significant differences when compared to the corresponding control plants in the same treatments at p<0.001. B. Target protein levels. Total protein isolated from the samples in (A) was electrophoresed on SDS-PAGE gels and blotted onto nitrocellulose membranes. A FLAG antiserum was used to detect the tagged target proteins during western analysis as described in the Materials and Methods (top panels). A duplicate gel was Coomassie-stained and used as a loading control (bottom panels). Similar results were obtained for two biological replicates. C. Diagrammatic representation of target mRNA cleavage sites determined using 5′RACE. Thick black lines represent open reading frames (nucleotide numbering begins at the start codon indicated by the black arrow), and the putative miRNA interaction site is shown as a gray box, with the nucleotide position within the ORF indicated. The nucleotides shown in red in each mRNA target are mismatches with the corresponding miRNA. The number of sequenced 5′RACE clones corresponding to each site is indicated by vertical arrowheads.

Mentions: We first performed qRT-PCR to check the mRNA levels of targets during co-expression (Fig. 5A). In the presence of slmiR482f, expression of its both putative target genes Solyc08g075630 and Solyc08g076000 were not significantly decreased. In contrast, slmiR5300 targets Solyc05g008650 and Solyc09g018220 were greatly suppressed by the presence of the miRNA; in the case of Solyc09g018220 transcript levels were reduced by almost 90% (Fig. 5A).


MicroRNAs suppress NB domain genes in tomato that confer resistance to Fusarium oxysporum.

Ouyang S, Park G, Atamian HS, Han CS, Stajich JE, Kaloshian I, Borkovich KA - PLoS Pathog. (2014)

Co-expression of slmiRNAs and predicted targets in Nicotiana benthamiana leaves.A. Levels of target mRNAs. qRT-PCR was used to determine relative levels of predicted tomato mRNAs in N. benthamiana leaves expressing only empty vector; target mRNA and a control miRNA (slmiR166); target mRNA and the appropriate miRNA (slmiR482f or slmiR5300); or target mRNA and empty vector. Values were normalized to N. benthamiana actin. Errors are expressed as the standard error. *** indicates significant differences when compared to the corresponding control plants in the same treatments at p<0.001. B. Target protein levels. Total protein isolated from the samples in (A) was electrophoresed on SDS-PAGE gels and blotted onto nitrocellulose membranes. A FLAG antiserum was used to detect the tagged target proteins during western analysis as described in the Materials and Methods (top panels). A duplicate gel was Coomassie-stained and used as a loading control (bottom panels). Similar results were obtained for two biological replicates. C. Diagrammatic representation of target mRNA cleavage sites determined using 5′RACE. Thick black lines represent open reading frames (nucleotide numbering begins at the start codon indicated by the black arrow), and the putative miRNA interaction site is shown as a gray box, with the nucleotide position within the ORF indicated. The nucleotides shown in red in each mRNA target are mismatches with the corresponding miRNA. The number of sequenced 5′RACE clones corresponding to each site is indicated by vertical arrowheads.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4199772&req=5

ppat-1004464-g005: Co-expression of slmiRNAs and predicted targets in Nicotiana benthamiana leaves.A. Levels of target mRNAs. qRT-PCR was used to determine relative levels of predicted tomato mRNAs in N. benthamiana leaves expressing only empty vector; target mRNA and a control miRNA (slmiR166); target mRNA and the appropriate miRNA (slmiR482f or slmiR5300); or target mRNA and empty vector. Values were normalized to N. benthamiana actin. Errors are expressed as the standard error. *** indicates significant differences when compared to the corresponding control plants in the same treatments at p<0.001. B. Target protein levels. Total protein isolated from the samples in (A) was electrophoresed on SDS-PAGE gels and blotted onto nitrocellulose membranes. A FLAG antiserum was used to detect the tagged target proteins during western analysis as described in the Materials and Methods (top panels). A duplicate gel was Coomassie-stained and used as a loading control (bottom panels). Similar results were obtained for two biological replicates. C. Diagrammatic representation of target mRNA cleavage sites determined using 5′RACE. Thick black lines represent open reading frames (nucleotide numbering begins at the start codon indicated by the black arrow), and the putative miRNA interaction site is shown as a gray box, with the nucleotide position within the ORF indicated. The nucleotides shown in red in each mRNA target are mismatches with the corresponding miRNA. The number of sequenced 5′RACE clones corresponding to each site is indicated by vertical arrowheads.
Mentions: We first performed qRT-PCR to check the mRNA levels of targets during co-expression (Fig. 5A). In the presence of slmiR482f, expression of its both putative target genes Solyc08g075630 and Solyc08g076000 were not significantly decreased. In contrast, slmiR5300 targets Solyc05g008650 and Solyc09g018220 were greatly suppressed by the presence of the miRNA; in the case of Solyc09g018220 transcript levels were reduced by almost 90% (Fig. 5A).

Bottom Line: One slmiR5300 target corresponds to tm-2, a susceptible allele of the Tomato Mosaic Virus resistance gene, supporting functions in immunity to a fungal pathogen.The observation that none of the targets correspond to I-2, the only known resistance (R) gene for F. oxysporum in tomato, supports roles for additional R genes in the immune response.Taken together, our findings suggest that Moneymaker is highly susceptible because its potential resistance is insufficiently expressed due to the action of miRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology and Microbiology, Institute for Integrative Genome Biology, University of California, Riverside, Riverside, California, United States of America.

ABSTRACT
MicroRNAs (miRNAs) suppress the transcriptional and post-transcriptional expression of genes in plants. Several miRNA families target genes encoding nucleotide-binding site-leucine-rich repeat (NB-LRR) plant innate immune receptors. The fungus Fusarium oxysporum f. sp. lycopersici causes vascular wilt disease in tomato. We explored a role for miRNAs in tomato defense against F. oxysporum using comparative miRNA profiling of susceptible (Moneymaker) and resistant (Motelle) tomato cultivars. slmiR482f and slmiR5300 were repressed during infection of Motelle with F. oxysporum. Two predicted mRNA targets each of slmiR482f and slmiR5300 exhibited increased expression in Motelle and the ability of these four targets to be regulated by the miRNAs was confirmed by co-expression in Nicotiana benthamiana. Silencing of the targets in the resistant Motelle cultivar revealed a role in fungal resistance for all four genes. All four targets encode proteins with full or partial nucleotide-binding (NB) domains. One slmiR5300 target corresponds to tm-2, a susceptible allele of the Tomato Mosaic Virus resistance gene, supporting functions in immunity to a fungal pathogen. The observation that none of the targets correspond to I-2, the only known resistance (R) gene for F. oxysporum in tomato, supports roles for additional R genes in the immune response. Taken together, our findings suggest that Moneymaker is highly susceptible because its potential resistance is insufficiently expressed due to the action of miRNAs.

Show MeSH
Related in: MedlinePlus