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Regulation of innate immunity to the fungal pathogen Fusarium oxysporum by microRNAs in tomato

Shouqiang Ouyang;Gyungsoon Park;Hagop Atamian;Cliff S.Han;Jason Stajich;Isgouhi Kaloshian;Katherine A.Borkovich

  MicroRNAs(miRNAs) play an important role as regulators of growth and development in plants.Several miRNA families target genes encoding disease resistance proteins,such as nucleotide binding site-leucine-rich repeat(NBS-LRR) plant innate immune receptors.The filamentous fungus Fusarium oxysporum f.sp.lycopersici causes vascular wilt disease in susceptible tomato(Solarium lycopersicum) plants.We explored a possible role for miRNAs in tomato defense against F.oxysporum using deep sequencing of small RNA libraries.Comparative miRNA profiling of sensitive(Moneymaker) and resistant(Motelle) tomato cultivars infected with F.oxysporum revealed several miRNAs that correlated with disease resistance.In particular,slmiR482 f and slmiR5300,two members of the miR482/2118 microRNA superfamily,were repressed during infection with F.oxysporum.Northern analysis confirmed that four of the predicted targets of these two miRNAs exhibited increased expression in the resistant Motelle cultivar and encode nucleotide-binding site(NBS) domain-containing proteins.Furthermore,the ability of two predicted mRNA targets of slmiR482f(Solyc08g075630 and Solyc08g076000) and two targets of slmiR5300(Solyc05g008650and Solyc09g018220) to be regulated by the miRNAs was confirmed by Agrobacterium-mediated transient co-expression in Nicotiana benthamiana.The results also revealed evidence for mRNA cleavage and/or translational inhibition mechanisms during regulation of target gene expression by the microRNAs.A TRV-based virus-induced gene silencing approach in the resistant Motelle cultivar revealed a clear role in resistance to F.oxysporum for one target of slmiR482f(Solyc08g075630;Fusarium Resistance Protein-1;FRP-1) and two targets of slmiR5300(Solyc05g008650;FRP-2 and Solyc09g018220;FRP-3).To our knowledge,this is the first demonstration of microRNAs that mediate resistance to F.oxysporum in tomato and first evidence of a role for miR5300 in pathogen resistance in any plant system.……