Isolation, Identification, And Functional Analysis Of Small RNAs Induced By Wheat Powdery Mildew(Blumeria Graminis F. Sp. Tirtici)

MicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between20and24nucleotides. They play a key role during plant development by negatively regulating gene expression atthe posttranscriptional level. Moreover, increasing evidence indicates that host small RNAs also play acritical role during the plant immune responses. Wheat is one of the most important crops with higheconomic value. More and more miRNAs have been discovered in wheat, but functions of thesemiRNAs on resistance to powdery mildew (PM) have rarely been disclosed. It is important to study themiRNAs roles in PM resistance which may facilitate more efficient strategy in wheat breeding forresistance to PM. Heat shock proteins90s (Hsp90s) are ubiquitous molecular chaperones in the cells ofeukaryta and eubacteria. They play key roles in signal transduction, protein folding, protein degradation,as well as growth and developmental programs. Cytosolic Hsp90proteins have also been found to playimportant roles in plant immune responses to pathogens. Pathogen effector-triggered immunity (ETI) inhigher plants is known to be mediated by intracellular immune receptors-R proteins. Many R proteinshave been found to be functionally dependent on cytosolic Hsp90s.In this study, to discover and analyze important small RNAs responsive to PM in wheat, wesequenced the wheat small RNAs libraries before and after Bgt inoculation. A total of38miRNAs wereobtained, including22known and16novel miRNAs. The sequencing results of miR396were repeatedby Northern blot, so we characterized the function of miR396, the main results are as following:⑴To discover miRNAs response to PM,6small RNA libraries using the young leaf of wheatinoculated with PM were constructed. The expression patterns of some miRNAs are confirmed bynorthern blot, miR396responses the same way under the two detection methods.⑵To comprehensively investigate small RNA targets, a degradomelibraries derived from themixture tissues of young leaf before and after Bgt inoculation of wheat was constructed and used forsequencing. Two hundreds and thirty-two genes targeted were detected in our degradome libraries. It isthe first time in wheat using degradome sequencing analysis of the target genes of miRNAs.⑶Degradome sequencing results showed that Hsp90is the target genes of miR396in wheat, andthis result is confirmed using5’ RACE, which confirms the result that TaHsp90.2and TaHsp90.3is thetarget genes of miR396.⑷To study functions of three classes of wheat Hsp90genes, virus-induced gene silencing system(VIGS) was used to analyze and the result showed that TaHsp90.2and TaHsp90.3also play importantroles in the wheat PM resistance pathway.Our studies discovered miRNAs responsive to PM by small RNA deep sequencing of librariesderived from tissues inoculated with PM. VIGS assays showed that miR396targets two Hsp genes thatare involved in PM resistance. Combined with previous studies that Hsp90genes play important roles inthe stripe rust resistance pathway of wheat, the function of the these genes and unraveled an miRNAmediated PM resistancec mechanism in wheat were expanded in this study.