Identification Of MicroRNA-like RNAs By High-throughput Sequencing And A Novel Cinnamyl Alcohol Dehydrogenase(CAD)-like Reductase In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum（Lib.）de Bary is a famous necrotrophic fungal pathogen with worldwide distribution,which able to produce sclerotia against various adverse environmental conditions.Therefore,sclerotial development is a crucial process in the lifecycle of S.sclerotiorum and blocking this process might be an efficient method to control Sclerotinia diseases in the field.MicroRNAs are small non-coding RNAs of approximately 22 nt with important regulatory roles in post-transcriptional regulation of gene expression,as now well known in animals and plants.Recently,mil RNAs have also been reported existing in filamentous fungi including Neurospora crassa.Here we used Solexa sequencing to obtain 13,229,401 high-quality reads,a total of 5,645,718 reads were mapped to the S.sclerotiorum genome corresponding to 348,108 unique s RNAs.Among the s RNAs,two mil RNAs and 42 mil RNA candidates were identified by sequence analysis.Northern blot and RT-PCR results showed that some mil RNAs and candidates are differentially expressed in sclerotial development suggesting the role of these mil RNAs and candidates in this biological process.Our results provide new insights into the functional roles of small RNAs and add new resources for the study of plant pathogenic fungi.To further investigate the mechanism of sclerotial development in S.sclerotiorum,gene expression profiles during hyphal and sclerotial growth were constructed using Illumina-Solexa sequencing.In this expression profile database,the gene SS1G₁0803（Gen Bank accession number: XM₀01588306.1）showed high expression level during sclerotial development compared to the hyphal growth stage.SS1G₁0803 contains three conserved domains in the Cinnamyl alcohol dehydrogenase（CAD）family,then phylogenetic analysis indicated that the homologs of CAD are widely present in fungi,but CADs in plant and fungi are obviously divided into two groups,indicating their different roles in plant and fungi.Cinnamyl alcohol dehydrogenase（CAD）catalyzes the last step of monolignol biosynthesis before oxidative polymerization in the cell wall.However,many CAD-like proteins have low CAD enzymatic activity and their actual biochemical roles remain unknown.Sclerotial development is a crucial process in the lifecycle for many plant fungal pathogens.The gene SS1G₁0803,named as Ss-CAD,is required for the normal sclerotial development of Sclerotinia sclerotiorum.Ss-CAD was significantly up-regulated at the initial stage of sclerotial development.Target silencing of Ss-CAD delayed the sclerotia formation and reduced sclerotia yield,but Ss-CAD silenced strains showed normal hyphal growth and virulence compared with the wild strain.The expression levels of ROS related genes,such as Nox1,Nox2 and Nox R,were down-regulated in the Ss-CAD silenced strains.Target silencing of Nox R in S.sclerotiorum showed the same phenotype in sclerotial formation with Ss-CAD silenced strains.Exogenous oxidants and NADPH could restore the sclerotial development.Sclerotial development of Ss-CAD silenced strains could also be restored by Nox1 over-expression.These results demonstrated that Ss-CAD is involved in the interaction with NADPH oxidases pathway to affect sclerotial development in S.sclerotiorum.