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

Abstract

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.

Reactive oxygen species and hormone signaling cascades in endophytic bacterium induced essential oil accumulation in Atractylodes lancea

“…The elongation factor 1a (EF1a) gene was used as the reference gene for normalization (Ren and Dai 2012). Quantification of the relative changes in the transcript levels of HMGR and DXR genes was performed using the comparative 2 -DDCt method (Liang et al 2015). The PCR protocol was 95°C for 5 min followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. The primers used in the study are listed in Table S1 (Wang et al 2015b).…”

Defense-related proteins involved in sugarcane responses to biotic stress

“…They are competitive and reversible inhibitors of cysteine proteases (Martínez and Díaz, 2008). The genes of the cystatin family have been identified and characterized in some plant species, demonstrating functions in defense against pathogens (Bobek and Levine, 1992; Gutierrez-Campos et al , 1999; Belenghi et al ., 2003), in response to insect attack (Goulet et al , 2008; Konrad et al , 2008; Liang et al , 2015), in programmed cell death (Solomon et al , 1999; Zhao et al , 2013), in seed germination (Hwang et al , 2009, Zhao et al , 2014), and in responses to abiotic environmental stresses (Hwang et al , 2010). Furthermore, the genes in the cystatin family were differentially expressed in response to different abiotic/biotic stresses, with essential roles in plant defense and hypersensitive cell death (Koiwa et al .…”

Phytocystatins: Defense Proteins against Phytophagous Insects and Acari

“…Microarray analysis of tomato responses to the spider mite Tetranychus urticae feeding revealed the up-regulation of a multicystatin as a defence protein [25]. The silkworm Bombyx mori induced the expression of five mulberry cystatin genes, being one of them stable to silkworm gut proteases [26]. Other lines of evidence have corroborated this putative defence role.…”