Biotic stress-induced expression of mulberry cystatins and identification of cystatin exhibiting stability to silkworm gut proteinases

Main conclusion Biotic stresses induce the expression of mulberry cystatins. MaCPI-4 protein is stable in silkworm digestive fluid and accumulates in gut food debris and frass. Plant cystatins are considered to be involved in defense responses to insect herbivores though little is known about how cystatins from the natural host respond to a specialist herbivory and the following postingestive interaction is also poorly understood. Here, we studied the biotic stress-mediated inductions of cystatins from mulberry tree, and examined the stability of mulberry cystatin proteins in the gut of silkworm, Bombyx mori , a specialist insect feeding on mulberry leaf. First, we cloned and characterized six cystatin genes from a mulberry cultivar, Morus atropurpurea Roxb., named as MaCPI - 1 to MaCPI - 6 . The recombinant MaCPI-1, MaCPI-3 and MaCPI-4 proteins, which showed inhibitory effects against papain in vitro, were produced. Silkworm herbivory as well as methyl jasmonate (MeJA) treatment induced the expression of five mulberry cystatin genes, and the highest inductions were observed from MaCPI - 1 and MaCPI - 6 . Mechanical wounding led to the inductions of four cystatin genes. The differential induction occurred in MaCPI - 2 . The induced protein changes were detected from three mulberry cystatins comprising MaCPI-1, MaCPI-3 and MaCPI-4. In vivo and in vitro assays showed that MaCPI-1 and MaCPI-3 proteins were susceptible to silkworm digestive fluid and MaCPI-4 had an antidigestive stability, and was detected in silkworm gut and frass. Collectively, our data indicated that biotic stresses resulted in the transcriptional inductions and protein changes of mulberry cystatins (MaCPIs), and identified MaCPI-4 with stability in the gut of its specialist herbivore.