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Supplementary Fig. 4 - Analysis of chitin-binding proteins from Manduca sexta provides new insights into evolution of peritrophin A-type chitin-binding domains in insects
Abstract
Phylogenetic analysis of the chitin-binding domains (CBDs) of PMPs from Coleoptera (square): Tribolium castaneum (Tc; green square), from Crustacea (reverse triangle): Daphnia pulex (Dp; dark blue reverse triangle), from Diptera (diamond): Culex quinquefasciatus (Cq; light blue diamond), from Hymenoptera (triangle): Nasonia vitripennis (Nv; brown triangle), and from Lepidoptera (circle): Bombyx mori (Bm; red circle), Danaus plexippus (Dpl; purple circle), Helicoverpa armigera (Ha; light green circle), Mamestra configurata (Mc, blue circle), M. sexta (Ms, white circle), Spodoptera litura (Sl; gray circle) and Trichoplusia ni (Tn; yellow circle). The accession numbers of all the proteins used are listed in Supplementary Table 1. The rank of the CBD in the PMP is indicated by a number (from 1 to 19) in brackets at the end of the PMP name.
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Microbial lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of crystalline polysaccharides including chitin and cellulose. The discovery of a large assortment of LPMO-like proteins widely distributed in insect genomes suggests that they could be involved in assisting chitin degradation in the exoskeleton, tracheae and peritrophic matrix during development. However, the physiological functions of insect LPMO-like proteins are still undetermined. To investigate the functions of insect LPMO subgroup I-like proteins, which contain an AA15 LPMO catalytic domain and a conserved C-terminal cysteine-rich motif, two evolutionarily distant species, Tribolium castaneum and Locusta migratoria, were chosen for study. RNAi for the T. castaneum protein, TcLPMO1, caused molting arrest at all developmental stages, whereas RNAi of the L. migratoria protein, LmLPMO1, prevented only adult eclosion. In both species, LPMO1-deficient animals were unable to shed their exuviae and died. TEM analysis revealed failure of turnover of chitinous cuticle, which is critical for completion of molting. Purified recombinant LPMO1-like protein from Ostrinia furnacalis (rOfLPMO1) exhibited oxidative cleavage activity and substrate preference for chitin. These results reveal for the first time the physiological importance of catalytically active LPMO1-like proteins from distant insect species and provide new insight into the enzymatic mechanism of chitin turnover during molting.
Alternative splicing is a common feature in eukaryotes that not only increases the transcript diversity, but also has have functional consequences. In insects, alternative splicing has been found associated with resistance to pesticides and Bt toxins. Up to date the alternative splicing in western corn rootworm (Diabrotica virgifera virgifera LeConte) has not been studied. To investigate its alternative splicing pattern and relation to Bt resistance, we carried out single‐molecule real‐time (SMRT) transcript sequencing and Iso‐seq analysis on resistant, eCry3.1Ab‐selected and susceptible, unselected, western corn rootworm neonate midguts which fed on seedling maize with and without eCry3.1Ab for 12 and 24 hours. We present transcriptome‐wide alternative splicing patterns of western corn rootworm midgut in response to feeding on eCry3.1Ab‐expressing corn using a comprehensive approach that combines both RNA‐seq and SMRT transcript sequencing techniques. The results showed genes in western corn rootworm are highly alternatively spliced, which happens on 67.73% of multi‐exon genes. One of the alternative splicing events we identified was a novel peritrophic matrix protein with two alternative splicing isoforms. Analysis of differential exon usage between resistant and susceptible colonies showed that in eCry3.1Ab‐resistant western corn rootworm, expression of one isoform was significantly higher than in the susceptible colony, while no significant differences between colonies were observed with the other isoform. Our results provide the first survey of alternative splicing in western corn rootworm and suggest that the observed alternatively spliced isoforms of peritrophic matrix protein may be associated with eCry3.1Ab resistance in western corn rootworm.
Chitin, the matrix polymer of arthropod exoskeleton, occurs in three forms that have different structural and mechanical properties. In the insect cuticle and the peritrophic matrix, individual chitin chains are further organized into higher order structures that give chitinous matrices their unique and widely differing properties such as rigidity, elasticity and waterproofing. In this chapter, we review the biochemical pathways of chitin biosynthesis, degradation and modification. In many cases, there are multiple isozymes for carrying out each step of chitin metabolism with specialization among members of families of isozymes. The gene families encoding the enzymes of chitin metabolism and their regulation are presented. The roles of chitin metabolism isoenzymes within families as revealed by gene deletion studies or RNA interference are discussed. How the association of chitin with different assortments of proteins modifies the properties of chitin matrices is briefly outlined.
'Candidatus Liberibacter asiaticus' (CLas), the bacterial pathogen associated with citrus greening disease, is transmitted by Diaphorina citri, the Asian citrus psyllid. Interactions among D. citri and its microbial endosymbionts, including 'Candidatus Profftella armatura', are likely to impact transmission of CLas. We used quantitative mass spectrometry to compare the proteomes of CLas(+) and CLas(-) populations of D. citri, and found that proteins involved in polyketide biosynthesis by the endosymbiont Profftella were up-regulated in CLas(+) insects. Mass spectrometry analysis of the Profftella polyketide diaphorin in D. citri metabolite extracts revealed the presence of a novel diaphorin-related polyketide and the ratio of these two polyketides was changed in CLas(+) insects. Insect proteins differentially expressed between CLas(+) and CLas(-) D. citri included defense and immunity proteins, proteins involved in energy storage and utilization, and proteins involved in endocytosis, cellular adhesion, and cytoskeletal remodeling which are associated with microbial invasion of host cells. Insight into the metabolic interdependence between the insect vector, its endosymbionts, and the citrus greening pathogen reveals novel opportunities for control of this disease, which is currently having a devastating impact on citrus production worldwide.
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