Thioredoxin peroxidase gene is involved in resistance to biocontrol fungus Nomuraea rileyi in Spodoptera litura: gene cloning, expression, localization and function.

Thioredoxin peroxidases (Tpxs) are a ubiquitous family of antioxidant enzymes that play important roles in protecting organisms against oxidative stress. Here, one Tpx was cloned from Spodoptera litura named as SlTpx. The full-length cDNA consists of 1165 bp with 588 bp open reading frame, encoding 195 amino acids. The putative amino acid sequence shared >70% identity with Tpxs from other insects. Phylogenetic analysis revealed that SlTpx is closely related to other available lepidopteran Tpxs. Real-time PCR analysis showed that SlTpx can be induced by Nomuraea rileyi infection in some detected tissues at the mRNA level. The strongest expression was found in hemocytes of unchallenged and N. rileyi-challenged S. litura. Western blotting showed SlTpx protein in the hemocytes, head and cuticle from normal S. litura. However, when N. rileyi was inoculated into the body cavity of S. litura larvae, SlTpx protein was detected in head, hemocytes, fatbody, midgut, malpighian tubule, but not in the hemolymph and cuticle. Moreover, time-course analysis showed that SlTpx mRNA/protein expression levels were up-regulated in the hemocytes, when S. litura were infected by N. rileyi or injected with H2O2. The levels of N. rileyi-induced reactive oxygen species (ROS) in hemocytes were evaluated, and revealed that N. rileyi infection caused generation of ROS, and induced changes in expression of SlTpx. In addition, the heterologously expressed protein of this gene in Escherichia coli showed antioxidant activity; it removed H2O2 and protected DNA. Knocking down SlTpx transcripts by dsRNA interference resulted in accelerated insect death with N. rileyi infection. This is believed to be the first report showing that SlTpx has a significant role in resisting oxidative stress caused by N. rileyi infection.

Chen H ，Yin Y ，Feng E ，Li Y ，Xie X ，Wang Z ... -  《-》

Structure and expression of a cysteine proteinase gene from Spodoptera litura and its response to biocontrol fungus Nomuraea rileyi.

Cysteine proteinases (Cyps) play vital roles in many biological processes, including physiological and pathological reactions. In the present study, we cloned a full cDNA of SlCyp, encoding a 344-amino-acid protein from Spodoptera litura. The putative amino acid sequence shared >75% identity with Cyps from other insects. A phylogenetic analysis revealed that SlCyp is closely related to other known lepidopteran Cyps. Real-time PCR and Western blotting analyses showed that SlCyp is induced by Nomuraea rileyi infection in all the tissues tested. The strongest SlCyp mRNA and protein expression was found in haemocytes, followed by the fat bodies, of unchallenged and N. rileyi-challenged S. litura. A time-course analysis showed that SlCyp mRNA and protein expression levels were upregulated in the haemocytes and fat bodies by N. rileyi infection. Upon N. rileyi infection, the proteolytic activities of SlCyp were also significantly higher in the haemolymph than in normal or phosphate-buffered-saline-challenged controls. These results suggest that SlCyp plays an important role in the innate immunity of S. litura in response to N. rileyi. SlCyp mRNA and protein expression and activities were also elevated during sixth-instar moulting and metamorphosis. Knocking down SlCyp transcripts with double-stranded RNA interference caused prepupal, pupal, and adult phenotypic changes, and SlCyp-silenced mutant larvae displayed a significantly lower survival rate after N. rileyi infection. These facts suggest that SlCyp plays a significant role in resisting N. rileyi infection and an essential role in larval development. Our data should facilitate the development of techniques for S. litura control.

Chen H ，Yin Y ，Feng E ，Xie X ，Wang Z ... -  《-》

Gene cloning, expression, and function analysis of SpL14-3-3ζ in Spodoptera litura and its response to the entomopathogenic fungus Nomuraea rileyi.

The 14-3-3 proteins, a highly evolutionarily conserved and ubiquitous protein family in eukaryotic cells, have a range of biological functions including regulation of signal transduction, stress response, apoptosis, and control of the cell cycle. To investigate the function of 14-3-3 in Spodoptera litura, the full length of 14-3-3ζ was cloned from S. litura on the basis of an expressed sequence tag of 14-3-3ζ from the S. litura fat body suppression subtractive hybridization library, and named SpL14-3-3ζ. SpL14-3-3ζ cDNA was 1196 bp with an open reading frame of 744 bp, encoding 247 amino acids. Multiple alignment analysis revealed the putative amino acids shared >80% homology with 14-3-3ζ from other organisms and shared typical conservative structures. Phylogenetic analysis confirmed SpL14-3-3ζ was closely related to other available Lepidoptera 14-3-3ζ. Real-time PCR analysis indicated SpL14-3-3ζ was expressed throughout the developmental stages of S. litura, with a relatively high expression level in pre-pupa, and was expressed constitutively in all examined tissues with relatively high levels in hemocytes and midgut. Moreover, the transcription level of SpL14-3-3ζ could be induced by Nomuraea rileyi infection, up-regulated in hemocytes, followed by head, fat body and midgut. Knocking down SpL14-3-3ζ transcripts by RNAi significantly increased S. litura sensitivity to fungal infection, and resulted in higher mortality of S. litura during the larval development. These results provide novel insights into the 14-3-3ζ signal regulation which may be related to host defense as well as larval development in S. litura.

Feng E ，Chen H ，Li Y ，Jiang W ，Wang Z ，Yin Y ... -  《-》

Identification and expression analysis of QM-like gene from Spodoptera litura after challenge by the entomopathogenic fungus Nomuraea rileyi.

A partial sequence of QM homologue was isolated from a Spodoptera litura fatbody suppression subtractive hybridization library. The full-length Spodoptera litura QM (SpLQM) cDNA of 838 bp contains a 5' untranslated region (UTR) of 112 bp, a 3' UTR of 66 bp, and an open reading frame of 660 nucleotides coding for a 219 amino acid peptide with a molecular weight of 25.5 kDa. Analysis of SpLQM sequence revealed the presence of characteristic motifs, including the ribosomal protein L10 signature and SH3-binding motif. Multiple alignment analysis revealed that SpLQM shares an overall identity of 57.1-99.1% with other members of QM family. Phylogenetic analysis confirmed that SpLQM is closely related to other insect QMs. Analysis of the tissue expression pattern showed that the SpLQM mRNA was expressed in all tissues tested, with highest levels measured in hemocytes, followed by fat bodies. Upon Nomuraea rileyi challenge, SpLQM showed significant upregulation in fat bodies and hemocytes, while slightly upregulation in midguts. The results suggest that SpLQM might play an important role in the innate immunity of S. litura in response to N. rileyi infection. SpLQM was also successfully overexpressed in Escherichia coli, and the recombinant fusion protein SpLQM-His has a molecular weight of 32 kDa.

Li Y ，Wang ZK ，Chen H ，Feng EY ，Yin YP ... -  《JOURNAL OF INSECT SCIENCE》

Identification and analysis of genes differentially expressed in the Spodoptera litura fat body in response to the biocontrol fungus, Nomuraea rileyi.

Nomuraea rileyi is an important pathogenic fungus that can successfully control Spodoptera litura. However, little is known on how S. litura responds to N. rileyi infection. A forward suppression subtractive hybridization (SSH) cDNA library was constructed from the S. litura fat body and the up-regulated genes were identified to isolate differentially expressed genes in response to N. rileyi. A total of 345/1175 random clones screened by cDNA array dot blotting were sequenced, resulting in 117 uniquely expressed sequence tags (ESTs). Potential functional genes were identified by BLAST searches and were categorized into seven groups associated with different biological processes based on the literature and gene ontologies. Among 117 genes, 74 had matches in the non-redundant (NR) protein database and were found to be involved in different biological processes, while 43 of the screened genes were classified to the "unknown function" gene group. Notably, only two genes had previously been reported in S. litura and most of the screened genes showed less similarity to known sequences based on BLASTn results, suggesting that 115 genes were found for the first time in S. litura. Semi-quantitative RT-PCR analysis of seven randomly selected genes revealed that most were differentially expressed after N. rileyi infection. qRT-PCR analysis confirmed that four genes (Hsp70, Hsp90, gallerimycin, and cysteine proteinase) were significantly up-regulated after N. rileyi infection. Taken together, the present study identified up-regulated S. litura genes in response to N. rileyi infection. Further investigations are needed to unravel the molecular mechanisms of the genes or proteins potentially involved in the S. litura innate immune defense against N. rileyi infection.

Chen H ，Yin Y ，Li Y ，Mahmud MS ，Wang Z ... -  《-》