Abstract
Genes for host-plant resistant do exist in cotton (Gossypium spp.) but improvement of cotton cultivars with resistance is difficult due to intensive breeding. Identifying molecular-genetic mechanisms associated with disease resistance can offer a new way to combat a serious threat such as Fusarium oxysporum f. sp. vasinfectum (FOV). Here, we captured and annotated “top-layer” of abundantly and specifically expressed cotton root small RNA (sRNA) including microRNA (miR) sequences during FOV pathogenesis using size-directed and adenylated linker-based sRNA cloning strategy. A total of 4116 candidate sRNA sequences with 16 to 30 nucleotide (nt) length were identified from four complementary DNA (cDNA) libraries of noninfected and FOV race 3-infected roots of susceptible (“11970”) versus resistant (“Mebane B-1”) cotton genotypes (G. hirsutum L.). The highest numbers of sRNA signatures were those with 19–24 nt long in all libraries, and interestingly, the number of sRNAs substantially increased during FOV infection in a resistant genotype, while it sharply decreased in a susceptible genotype. In BLAST analysis, more than 73 % of sRNAs matched Gossypium (G. arboretum L., G. hirsutum, and G. barbadense L.) ESTs. A small percentage of sRNAs matched A. thaliana (1.68 %), T. cacao (1.26 %), fungal (2 %), and other organism (21.33 %) ESTs. mirBase comparisons showed that 4 % of sRNAs were homologous to previously reported plant miRs, among which we predicted novel cotton Ghr-miR-160 that was not registered in the cotton miR database. These major representative sRNA signatures targeted proteins associated with the key biological processes and molecular functions, explaining the molecular mechanisms of the host defense response during the FOV pathogenesis in cotton.
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Funding
This project was funded by the Office of International Research Programs (OIRP) of the United States Department of Agriculture (USDA)—Agricultural Research Service (ARS) in the frame of USDA-ARS—Former Soviet Union (FSU) cooperation programs with the research grant of UZB2-31016-TA-09. We acknowledge the Academy of sciences of Uzbekistan and US Civilian Research & Development Foundation (CRDF) for project coordination. Mention of trade names or commercial products in this article is solely for providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. The US Department of Agriculture is an equal opportunity provider and employer.
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Supplementary Fig. S1
sRNA isolation experimental procedure (A-D) and an example of concatemerazed sRNAs sequencing (E) chromatography: (A) - 15 % denaturing PAG electrophoresis of total RNAs isolated from infected (1, 2) and non-infected tissues (3, 4), (a)- mRNA/rRNA/tRNA fraction, (b)-small RNA fraction, M – low molecular weight molecular marker of 10 nt; (B) - 15 % denaturing PAG electrophoresis for 3′ linkering reaction where upper arrow shows 3′linkered product, while lower arrow shows a unlinkered small RNAs, (S) - 21 nt spike RNA; (C) 2 % agarose gel of RT-PCR after 5 and 3′ linkering reactions, (M)-50 bp DNA ladder; and (D) 2 % agarose gel for the colony PCR of concatemerazed sRNA products that were sub-cloned into TOPO-TA vector where (N) - negative PCR (from empty vector) amplification, (P) - positive clones, and (M)-50 bp DNA ladder. (PDF 751 kb)
Supplementary Fig. S2
GO analysis for biological process (upper) and molecular function (lower) on sRNAs targeted genes (%) from four libraries. (PPT 743 kb)
Supplementary Table S1
Detailed list of sRNAs cloned from FOV infected vs. non-infected cotton tissues and BLAST analysis results. (XLS 277 kb)
Supplementary Table S2
Target identification of sRNAs cloned from FOV infected vs. non-infected cotton tissues. (XLS 85 kb)
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Shapulatov, U.M., Buriev, Z.T., Ulloa, M. et al. Characterization of Small RNAs and Their Targets from Fusarium oxysporum Infected and Noninfected Cotton Root Tissues. Plant Mol Biol Rep 34, 698–706 (2016). https://doi.org/10.1007/s11105-015-0945-z
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DOI: https://doi.org/10.1007/s11105-015-0945-z