WSSV-responsive gene expression under the influence of PmVRP15 suppression

Abstract The viral responsive protein 15 from black tiger shrimp Penaeus monodon ( Pm VRP15), is highly up-regulated and produced in the hemocytes of shrimp with white spot syndrome virus (WSSV) infection. To investigate the differential expression of genes from P. monodon hemocytes that are involved in WSSV infection under the influence of Pm VRP15 expression, suppression subtractive hybridization (SSH) of Pm VRP15-silenced shrimp infected with WSSV was performed. The 189 cDNA clones of the forward library were generated by subtracting the cDNAs from WSSV-infected and Pm VRP15 knockdown shrimp with cDNAs from WSSV-infected and GFP knockdown shrimp. For the opposite subtraction, the 176 cDNA clones in the reverse library was an alternative set of genes in WSSV-infected shrimp hemocytes in the presence of Pm VRP15 expression. The abundant genes in forward SSH library had a defense/homeostasis of 26%, energy/metabolism of 23% and in the reverse SSH library a hypothetical protein with unknown function was found (30%). The differential expressed immune-related genes from each library were selected for expression analysis using qRT-PCR. All selected genes from the forward library showed high up-regulation in the WSSV-challenged Pm VRP15 knockdown group as expected. Interestingly, Pm HHAP, a hemocyte homeostasis associated protein, and granulin-like protein, a conserved growth factor, are extremely up-regulated in the absence of Pm VRP15 expression in WSSV-infected shrimp. Only transcript level of transglutaminase II, that functions in regulating hematopoietic tissue differentiation and inhibits mature hemocyte production in shrimp, was obviously down-regulated as observed from SSH results. Taken together, our results suggest that Pm VRP15 might have a function relevant to hemocyte homeostasis during WSSV infection.