Isolation and characterization of small RNAs from Fusarium wilt infected cotton root tissues

Functional Genomics

poster

Shapulatov, Umidjon; Buriev, Zabardast ; Ayubov, Mirzakamol; Norov, Tohir ; Saha, Sukumar; Ulloa, Mauricio ; Devor, Eric; Abdurakhmonov, Ibrokhim

Shapulatov, Umidjon

Abdurakhmonov, Ibrokhim

Along with many other biological and environmental threats, one of the main biological threats in cotton production is a devastating and aggressive wilting disease known as Fusarium wilt caused by the fungus Fusarium oxysporum f.sp. vasinfectum (FOV). Molecular mechanisms associated with Fusarium wilt resistance of Upland cotton (Gossypium hirsutum L.) are largely unknown. Because small RNAs and microRNAs play an important role in plant defense we characterized small RNA profiles during FOV race 3 pathogenesis in cotton. Four separate small RNA libraries (sRNAs) were prepared from cotton (G. hirsutum) roots (infected and uninfected), including FOV resistant Mebane B-1 line and FOV susceptible № 11970 line. We sequenced 4179 clones and detected 4116 small RNAs sequences of 16-30nt. Although the total number of unique tags were approximately the same among libraries (except № 11970 FOV susceptible infected line), these tags were substantially different based on the length distribution. However, the highest percentage of these unique sequence signatures was 21nt in length in all of the libraries. The Mebane B-1 line had the highest number of unique 21nt sequence signatures and the uninfected library had a greater number than its infected counterpart library suggesting the distribution of 21nt size sRNAs is different between these two lines depending upon FOV infection. BLAST results show that there are several kinds of RNA fragments such as mRNAs, rRNAs and other unknown short fragments. Greater than 73% of unique sRNAs from four libraries matched to Gossypium L. (G. arboreum, G. hirsutum, and G. barbadense) expressed sequence tags (ESTs). A small percentage of unique sRNAs matched to A.thaliana (1.68%), T. cacao (1.26%), Fungal (2%), and other organism (21.33%) ESTs. MirBase database search detected 4% of unique sRNAs to be homologous to some plant microRNAs described in the literature such as miR156i-3p, miR160a, miR169n-3p, miR171d-5p, miR172a, miR390a-3p, miR398b-5p, miR395l-5p, miR1069-3p, miR2949a-5p, miR2911, miR2916, miR166m-5p, and miR3476-5p. Our results showed only two sequence tags aligning with known plant miRNAs not registered in the cotton database yet. Mir-160, which was sequenced from FOV uninfected Mabane-1 young root tissues, is highly conserved in plants and mir-2911, which was seen in all four small RNA libraries, is known from Nicotiana tabacum and Populus euthratica databases. Analysis of target proteins from small RNA signatures detected important cellular components helping to explain the FOV pathogenesis regulation in cotton. Small RNA and microRNA sequence signatures characterized in this work will be helpful for developing innovative biotechnology tools to improve FOV resistance of cotton cultivars.