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Pair of MicroRNAs Plays Role in Tomato Resistance to Fungal Infection

NEW YORK (GenomeWeb) – Researchers have identified two microRNAs that are down-regulated in a tomato plant that is resistant to a fungal infection, but not in a tomato plant that is susceptible to the fungus.

By comparing miRNA levels in two closely related tomato cultivars — Moneymaker, which is susceptible to the Fusarium oxysporum fungus, and Motelle, which is not — researchers led by the University of California, Riverside's Katherine Borkovitch homed in on a number of miRNA whose levels varied between the cultivars upon infection.

Through an in silico approach, they further identified four possible targets for those miRNAs, and most of those targets were upregulated in Motelle, but not Moneymaker, upon infection, as they reported in PLOS Pathogens today.

"Our study provides a platform for differentially expressed miRNAs in tomato after F. oxysporum infection and demonstrates that plant miRNAs are involved in defense against F. oxysporum," Borkovitch and he colleagues said in their paper

There are more than 120 types of F. oxysporum that live in the soil and can cause disease in vegetable, fruit, and ornamental plants. The F. oxysporum f. sp. lycopersici the researchers focused on here attacks vascular bundles in its host plant, leading it to wilt.

MiRNAs, the researchers noted, have been linked to pathogen response in plants, including tomato, and here they searched for ones that were either upregulated in Moneymaker or downregulated in Motelle upon infection with F. oxysporum.

To do so, they generated four libraries for sequencing: Moneymaker treated with water, Moneymaker treated with F. oxysporum, Motelle treated with water, and Motelle treated with F. oxysporum. After Illumina sequencing of the libraries, the researchers uncovered 18 miRNAs associated with plant disease resistance, stress response, or transcription factors.

Northern blot analysis found that only two of those miRNAs — slmiR482f and slmiR5300 — decreased in Motelle after infection, indicating that these miRNA might regulate tomato plant defense genes.

Using the psRNANTarget algorithm, Borkovitch and her colleagues identified putative binding sites for slmiR482f and slmiR5300. The top hits, they noted, all contained full or partial nucleotide-binding domains. One slmiR5300 target, they added, corresponds to tm-2, a tomato mosaic virus susceptibility allele, but none of the targets corresponded to l-2, the only known F. oxysporum resistance gene in tomato.

After examining the levels of the possible mRNA targets in Motelle and Moneymaker treated with water or F. oxysporum using qRT-PCR, the researchers found that slmiR482f target Soly08g075630 was induced in Motelle, but not Moneymaker after fungal infections, as were slmiR5300 putative targets Solyc08g008650 and Solyc09g018220.

The slmiR482f target Solyc08g076000 mRNA levels didn't vary in either Motelle or Moneymaker upon infection, though its levels were generally higher in Motelle.

This, the researchers said, suggests that at least three of the predicted targets are regulated at the mRNA level by their respective miRNAs.

Through co-expression studies in which putative target protein genes and miRNA genes were both expressed in Nicotiana benthamiana leaves, the researchers reported that the target protein levels declined when the targets were expressed in conjunction with their corresponding miRNA, suggesting that slmiR482f and slmiR5300 are responsible for the downregulation.

Borkovitch and her colleagues also explored how these miRNAs might contribute to fungal resistance. Using a TRV-based virus-induced silencing system, they downregulated the target mRNA gene in Motelle, the cultivar that is resistant to F. oxysporum.

Control Motelle plants and those inoculated with empty vectors didn't exhibit symptoms when infected with F. oxysporum, while control Moneymaker plants did.

Further, Motelle plants with silenced genes did experience leaf wilting and discoloration, though their symptoms weren't as extreme as the control Moneymaker plants.

"Taken together, our findings suggest that Moneymaker is highly susceptible [to F. oxysporum infection] because its potential resistance is insufficiently expressed due to the action of miRNAs," Borkovitch and her colleagues said.