ANALYSIS OF TWO SUBTRACTION CDNA LIBRARIES HIGHLIGHTS SEVERAL HIGHLY ABUNDANT HETERODERA GLYCINES TRANSCRIPTS THAT ENCODE SMALL PROTEINS OF UNKNOWN FUNCTION WITH PUTATIVE 5´ ER SIGNAL PEPTIDES.

Authors: Tucker, Mark; XUE, PING - UNIVERSITY OF MARYLAND; Raina, Anjana; Ehrenfried, Mindy; Thai, Vanessa

Submitted to: Journal of Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/30/2005
Publication Date: 12/30/2005
Citation: Tucker, M.L., Xue, P., Raina, A., Ehrenfried, M.L., Thai, V.K. 2005. Analysis of two subtraction cdna libraries highlights several highly abundant heterodera glycines transcripts that encode small proteins of unknown function with putative 5´ er signal peptides. Journal of Nematology. 37:422-428.

Interpretive Summary: Soybean cyst nematode (SCN) is the most economically damaging pathogen of soybean causing an estimated annual loss of one billion dollars to the soybean crop in the USA. Identifying new mechanisms for both natural and engineered resistance is essential to reduce current losses and maintain resistance to aggressive strains of nematodes that continuously evolve new mechanisms to evade plant defenses. We have used a technique called subtractive cloning to create two gene libraries that are enriched for gene transcripts that are specifically expressed during nematode infection. One of the two gene libraries was made using root extracts that included developing nematodes at later stages of their life cycle. Half of the gene transcripts in this late library were of nematode origin. Many of these transcripts encoded small proteins with sequence at the beginning of the protein that is used for secretion of the protein from the nematode into the soybean root. If secreted into roots, these proteins could act as signals that alter functions in the cells of the soybean root. Characterization of these nematode genes and the encoded proteins will aid in our understanding of how the nematode interacts with the host cell to cause the marked changes in root cell morphology and thus provide a tool for disrupting the ability of the nematode to infect soybean roots. Information of this kind will aid scientists and industrial partners in the development of new strategies to create and maintain resistance to SCN.

Technical Abstract: We constructed two subtraction libraries from RNA extracted at early and late stages in the development of soybean cyst nematodes (SCN), Heterodera glycines, in soybean roots. The cDNAs from inoculated roots were subtracted with cDNAs prepared from un-inoculated roots and SCN eggs. Currently, 384 clones from each library have been sequenced and arrayed onto nylon membranes. BLAST searches revealed that 191 (50%) of the cDNAs in the late library were of nematode origin. Alignment of the 191 sequences produced 28 contigs and 1 singlet. Six SCN consensus sequences were selected for further study because they were abundantly represented in the subtraction library and produced strong hybridization signals on membrane arrays. All six sequences included open reading frames with a signal peptide at the 5´ ends. Four of the six encode predicted peptides smaller than 7 kDa and the other two 13.2 and 32.6 kDa. RNA blot hybridization and 5´ RACE confirmed that the derived consensus sequences were full-length or nearly full-length. Moreover, only two of the six transcripts had similarity with ESTs from other nematodes, and these two had similarity with a total of only 4 ESTs, which suggests a specialized role for these peptides in H. glycines development.