Plant latex and other exudates as plant defense systems: roles of various defense chemicals and proteins contained therein

Plant latex and other exudates are saps that are exuded from the points of plant damage caused either mechanically or by insect herbivory. Although many (ca. 10%) of plant species exude latex or exudates, and although the defensive roles of plant latex against herbivorous insects have long been suggested by several studies, the detailed roles and functions of various latex ingredients, proteins and chemicals, in anti-herbivore plant defenses have not been well documented despite the wide occurrence of latex in the plant kingdom. Recently, however, substantial progress has been made. Several latex proteins, including cysteine proteases and chitin-related proteins, have been shown to play important defensive roles against insect herbivory. In the mulberry (Morus spp.)-silkworm (Bombyx mori) interaction, an old and well-known model system of plant-insect interaction, plant latex and its ingredients - sugar-mimic alkaloids and defense protein MLX56 - are found to play key roles. Complicated molecular interactions between Apocynaceae species and its specialist herbivores, in which cardenolides and defense proteins in latex play key roles, are becoming more and more evident. Emerging observations suggested that plant latex, analogous to animal venom, is a treasury of useful defense proteins and chemicals that has evolved through interspecific interactions. On the other hand, specialist herbivores developed sophisticated adaptations, either molecular, physiological, or behavioral, against latex-borne defenses. The existence of various adaptations in specialist herbivores itself is evidence that latex and its ingredients function as defenses at least against generalists. Here, we review molecular and structural mechanisms, ecological roles, and evolutionary aspects of plant latex as a general defense against insect herbivory and we discuss, from recent studies, the unique characteristics of latex-borne defense systems as transport systems of defense substances are discussed based on recent studies.     

The green peach aphid,Myzus persicae,acquires a LIPOXYGENASE5-derived oxylipin from Arabidopsis thaliana,which promotes colonization of the host plant

Oxylipins derived from lipoxygenase (LOX) activity play important roles in plant growth, development and stress response. In a recent study, we provided evidence that infestation of Arabidopsis thaliana foliage by the green peach aphid (GPA; Myzus persicae), a phloem sap-consuming insect, was promoted by plant LOX5-derived oxylipins. In comparison to the wild-type (WT) plant, GPA population was smaller on the Arabidopsis lox5 mutant. The insect spent less time feeding from the sieve element and xylem of the lox5 mutant compared with the WT plant. In addition, compared with insects feeding on the WT plant, when on the lox5 mutant, the GPA was unable to suppress an antibiotic activity that is present in Arabidopsis vascular sap. Roots are the critical source of a LOX5-derived oxylipin(s) that promotes colonization of the foliage by GPA. Here we show that the 9-hydoxy-10E, 12Z-octadecadienoic acid (9-HOD), a LOX5-derived oxylipin, accumulated in GPA that were reared on the WT, but not the lox5 mutant plant. However, 9-HOD accumulated in insects reared on lox5 mutant plants that were irrigated with 9-HOD, thus indicating that the insect ingests oxylipins from the host plant. We further demonstrate that the host plant requires LOX5 function to promote expression of the defense regulatory gene PHYTOALEXIN-DEFICIENT4 in the foliage. Taken together, our previous observations and results presented here indicate that while the host plant utilizes LOX5-dependent factors for promoting defense mechanisms, GPA has evolved to utilize plant 9-LOX-derived oxylipins as cues to facilitate infestation, thus suggesting a complex involvement of oxylipins in Arabidopsis interaction with GPA.     

Comprehensive proteome analysis of lettuce latex using multidimensional protein-identification technology

Commercially, lettuce (Lactuca sativa) is one of the most important leafy vegetables. Lettuce produces a milky latex of variable chemical compositions within its laticifers. As a step toward understanding the main physiological roles of this latex in higher plants, we embarked on its proteomic analysis. We investigated 587 latex proteins that were identified from the lettuce latex using multidimensional protein-identification technology. A bioinformatics analysis showed that the most frequently encountered proteins in the latex were organellar proteins from plastids and mitochondria, followed by nucleic and cytoplasmic proteins. Functional classification of the identified proteins showed that proteins related to metabolism, cell rescue, defense, and virulence were the most abundant in lettuce latex. Furthermore, numerous resistance proteins of lettuce and viral proteins were present in the latex suggesting for the first time a possible function of the lettuce latex in defense or pathogenesis. To the knowledge of the authors, this is the first large-scale proteome analysis of lettuce latex.     

Shotgun analysis on the peritrophic membrane of the silkworm Bombyx mori

The insect midgut epithelium is generally lined with a unique chitin and protein structure, the peritrophic membrane (PM), which facilitates food digestion and protects the gut epithelium. We used gel electrophoresis and mass spectrometry to identify the extracted proteins from the silkworm PM to obtain an in-depth understanding of the biological function of the silkworm PM components. A total of 305 proteins, with molecular weights ranging from 8.02 kDa to 788.52 kDa and the isoelectric points ranging from 3.39 to 12.91, were successfully identified. We also found several major classes of PM proteins, i.e. PM chitin-binding protein, invertebrate intestinal mucin, and chitin deacetylase. The protein profile provides a basis for further study of the physiological events in the PM of Bombyx mori. [BMB Reports 2012; 45(11): 665-670]     

An immune-induced reeler protein is involved in the Bombyx mori melanization cascade

In this study, we isolated two reeler cDNAs from bacteria-challenged larval fat bodies of the silkworm, Bombyx mori. A reeler domain spanned most of the coding regions of these two cDNAs, and their expression patterns were different in B. mori larval tissues. The reeler1 gene was strongly induced by Escherichia coli K12 and Bacillus subtilis in B. mori larval hemocytes, fat bodies and midguts, but reeler2 was expressed at extremely low levels in these tissues. We focused on the reeler1 gene for functional analysis. Interference by double-stranded reeler1 RNA in vivo led to reduced nodule formation in bacteria-injected larvae, while the injection of recombinant Reeler1 promoted nodule formation in reeler1 gene-silenced larvae, indicating that Reeler1 is involved in the nodulation response. Knockdown of the reeler1 gene significantly decreased phenoloxidase activity in bacteria-challenged larval hemolymph, while injection of recombinant Reeler1 enhanced phenoloxidase activity, suggesting that Reeler1 is involved in the prophenoloxidase activation cascade. Our results provide new mechanistic evidence about the melanization cascade in the insect immunity.     

Sm2, a paralog of the Trichoderma cerato-platanin elicitor Sm1, is also highly important for plant protection conferred by the fungal-root interaction of Trichoderma with maize

Background

The proteins Sm1 and Sm2 from the biocontrol fungus Trichoderma virens belong to the cerato-platanin protein family. Members of this family are small, secreted proteins that are abundantly produced by filamentous fungi with all types of life-styles. Some species of the fungal genus Trichoderma are considered as biocontrol fungi because they are mycoparasites and are also able to directly interact with plants, thereby stimulating plant defense responses. It was previously shown that the cerato-platanin protein Sm1 from T. virens - and to a lesser extent its homologue Epl1 from Trichoderma atroviride - induce plant defense responses. The plant protection potential of other members of the cerato-platanin protein family in Trichoderma, however, has not yet been investigated.

Results

In order to analyze the function of the cerato-platanin protein Sm2, sm1 and sm2 knockout strains were generated and characterized. The effect of the lack of Sm1 and Sm2 in T. virens on inducing systemic resistance in maize seedlings, challenged with the plant pathogen Cochliobolus heterostrophus, was tested. These plant experiments were also performed with T. atroviride epl1 and epl2 knockout strains. In our plant-pathogen system T. virens was a more effective plant protectant than T. atroviride and the results with both Trichoderma species showed concordantly that the level of plant protection was more strongly reduced in plants treated with the sm2/epl2 knockout strains than with sm1/epl1 knockout strains.

Conclusions

Although the cerato-platanin genes sm1/epl1 are more abundantly expressed than sm2/epl2 during fungal growth, Sm2/Epl2 are, interestingly, more important than Sm1/Epl1 for the promotion of plant protection conferred by Trichoderma in the maize-C. heterostrophus pathosystem.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-014-0333-0) contains supplementary material, which is available to authorized users.     

in silico identification of protein-protein interactions in Silkworm,Bombyx mori

The Domesticated silkworm, Bombyx mori, an economically important insect has been used as a lepidopteran molecular model next only to Drosophila. Compared to the genomic information in silkworm, the protein-protein interaction data are limited. Therefore experimentally identified PPI maps from five model organisms such as E.coli, C.elegans, D.melanogaster, H. sapiens, S. cerevisiae were used to infer the PPI network of silkworm using the well-recognized Interlog based method. Among the 14623 silkworm proteins, 7736 protein-protein interaction pairs were predicted which include 2700 unique proteins of the silkworms. Using the iPfam interaction domains and the gene expression data, these predictions were validated. In that 625 PPI pairs of predicted network were associated with the iPfam domain-domain interactions and the random network has average of 9. In the gene expression method, the average PCC value of the predicted network and random network was 0.29 and 0.23100±0.00042 respectively. It reveals that the predicted PPI networks of silkworm are highly significant and reliable. This is the first PPI network for the silkworm which will provide a framework for deciphering the cellular processes governing key metabolic pathways in the silkworm, Bombyx mori and available at SilkPPI (http://210.212.197.30/SilkPPI/).     

An S-locus receptor-like kinase plays a role as a negative regulator in plant defense responses

Plant cells often use cell surface receptors to sense environmental changes and then transduce external signals via activated signaling pathways to trigger adaptive responses. In Arabidopsis, the receptor-like protein kinase (RLK) gene family contains more than 600 members, and some of these are induced by pathogen infection, suggesting a possible role in plant defense responses. We previously characterized an S-locus RLK (CBRLK1) at the biochemical level. In this study, we examined the physiological function of CBRLK1 in defense responses. CBRLK1 mutant and CBRLK1-overexpressing transgenic plants showed enhanced and reduced resistance against a virulent bacterial pathogen, respectively. The altered pathogen resistances of the mutant and overexpressing transgenic plants were associated with increased and reduced induction of the pathogenesis-related gene PR1, respectively. These results suggest that CBRLK1 plays a negative role in the disease resistance signaling pathway in Arabidopsis.     

Increased Insect Virulence in Beauveria bassiana Strains Overexpressing an Engineered Chitinase

Entomopathogenic fungi are currently being used for the control of several insect pests as alternatives or supplements to chemical insecticides. Improvements in virulence and speed of kill can be achieved by understanding the mechanisms of fungal pathogenesis and genetically modifying targeted genes, thus improving the commercial efficacy of these biocontrol agents. Entomopathogenic fungi, such as Beauveria bassiana, penetrate the insect cuticle utilizing a plethora of hydrolytic enzymes, including chitinases, which are important virulence factors. Two chitinases (Bbchit1 and Bbchit2) have previously been characterized in B. bassiana, neither of which possesses chitin-binding domains. Here we report the construction and characterization of several B. bassiana hybrid chitinases where the chitinase Bbchit1 was fused to chitin-binding domains derived from plant, bacterial, or insect sources. A hybrid chitinase containing the chitin-binding domain (BmChBD) from the silkworm Bombyx mori chitinase fused to Bbchit1 showed the greatest ability to bind to chitin compared to other hybrid chitinases. This hybrid chitinase gene (Bbchit1-BmChBD) was then placed under the control of a fungal constitutive promoter (gpd-Bbchit1-BmChBD) and transformed into B. bassiana. Insect bioassays showed a 23% reduction in time to death in the transformant compared to the wild-type fungus. This transformant also showed greater virulence than another construct (gpd-Bbchit1) with the same constitutive promoter but lacking the chitin-binding domain. We utilized a strategy where genetic components of the host insect can be incorporated into the fungal pathogen in order to increase host cuticle penetration ability.     

Enhancement of Larval RNAi Efficiency by Over-expressing Argonaute2 in Bombyx mori

RNA interference has been described as a powerful genetic tool for gene functional analysis and a promising approach for pest management. However, RNAi efficiency varies significantly among insect species due to distinct RNAi machineries. Lepidopteran insects include a large number of pests as well as model insects, such as the silkworm, Bombyx mori. However, only limited success of in vivo RNAi has been reported in lepidoptera, particularly during the larval stages when the worms feed the most and do the most harm to the host plant. Enhancing the efficiency of larval RNAi in lepidoptera is urgently needed to develop RNAi-based pest management strategies. In the present study, we investigate the function of the conserved RNAi core factor, Argonaute2 (Ago2), in mediating B. mori RNAi efficiency. We demonstrate that introducing BmAgo2 dsRNA inhibits the RNAi response in both BmN cells and embryos. Furthermore, we establish several transgenic silkworm lines to assess the roles of BmAgo2 in larval RNAi. Over-expressing BmAgo2 significantly facilitated both dsRNA-mediated larval RNAi when targeting DsRed using dsRNA injection and shRNA-mediated larval RNAi when targeting BmBlos2 using transgenic shRNA expression. Our results show that BmAgo2 is involved in RNAi in B. mori and provides a promising approach for improving larval RNAi efficiency in B. mori and in lepidopteran insects in general.     

A Brassica juncea chitinase with two-chitin binding domains show anti-microbial properties against phytopathogens and Gram-negative bacteria

In our recent paper in the Journal of Experimental Botany, we demonstrated that Brassica juncea BjCHI1 shows anti-fungal properties against phytopathogens, Colletotrichum truncatum, C. acutatum, Botrytis cinerea and Ascochyta rabiei. Furthermore, BjCHI1 which is an unusual plant chitinase with two (almost identical) chitin-binding domains, agglutinates Gram-negative bacteria, adversely affecting their growth. In contrast, BjCHI1 derivatives lacking one or both domains do not show agglutination activity, suggesting that both chitin-binding domains are essential for agglutination. Observations that agglutination could be relieved by addition of galactose, glucose or lactose, imply that BjCHI1 interacts with the carbohydrate components of the Gram-negative bacterial cell wall. We propose here, a model for BjCHI1-mediated agglutination between Gram-negative bacteria, through interaction of their adjacent cell walls mediated by the two chitin-binding domains of BjCHI1. BjCHI1 is a plant chitinase which has evolved towards acquiring an enhanced role in plant defense against fungi and Gram-negative bacteria. Hence, it is a promising candidate for applications against phytopathogens in plant genetic engineering via nuclear or plastid transformation.Key words: bacterial agglutination, chitin-binding domain, Indian mustard, lectin, phytopathogens, Pichia-expressed proteins, transplastomic tobacco     

Tobacco-expressed Brassica juncea chitinase BjCHI1 shows antifungal activity in vitro

We have previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains, and have shown that its mRNA is induced by wounding and methyl jasmonate treatment (K.-J. Zhao and M.-L. Chye, Plant Mol. Biol. 40 (1999) 1009–1018). By the presence of two chitin-binding domains, BjCHI1 resembles the precursor of UDA (Urtica dioica agglutinin) but, unlike UDA, BjCHI1 retains its chitinase catalytic domain after post-translational processing. Here, we indicate the role of BjCHI1 in plant defense by demonstrating its mRNA induction upon Aspergillus niger infection or caterpillar Pieris rapae (L.) feeding. To further investigate the biological properties of BjCHI1, we transformed tobacco with a construct expressing the BjCHI1 cDNA from the CaMV 35S promoter. Subsequently, we purified BjCHI1 from the resultant transgenic R₀ plants using a regenerated chitin column followed by fast protein liquid chromatography (FPLC). Also, the significance of the second chitin-binding domain in BjCHI1 was investigated by raising transgenic tobacco plants expressing BjCHI2, a deletion derivative of BjCHI1 lacking one chitin-binding domain. Colorimetric chitinase assays at 25 °C, pH 5, showed no significant differences between the activities of BjCHI1 and BjCHI2, suggesting that chitinase activity, due to the catalytic domain, is not enhanced by the presence of a second chitin-binding domain. Both BjCHI1 and BjCHI2 show in vitro anti-fungal activity toward Trichoderma viride, causing reductions in hyphal diameter, hyphal branching and conidia size.     

Identification and characterization of a novel chitinase-like gene cluster (AgCht5) possibly derived from tandem duplications in the African malaria mosquito, Anopheles gambiae

Insect chitinase 5 (Cht5), a well-characterized enzyme found in the molting fluid and/or integument, is classified as a group I chitinase and is usually encoded by a single gene. In this study, a Cht5 gene cluster consisting of five different chitinase-like genes (AgCht5-1, AgCht5-2, AgCht5-3, AgCht5-4 and AgCht5-5) was identified by a bioinformatics search of the genome of Anopheles gambiae. The gene models were confirmed by cloning and sequencing of the corresponding cDNAs and gene expression profiles during insect development were determined. All of these genes are found in a single cluster on chromosome 2R. Their open reading frames (ORF) range from 1227 to 1713 bp capable of encoding putative proteins ranging in size from 409 to 571 amino acids. The identities of their cDNA sequences range from 52 to 66%, and the identities of their deduced amino acid sequences range from 38 to 53%. There are four introns for AgCht5-1, two for AgCht5-2 and AgCht5-3, only one for AgCht5-4, but none for AgCht5-5 in the genome. All five chitinase-like proteins possess a catalytic domain with all of the conserved sequence motifs, but only AgCht5-1 has a chitin-binding domain. Phylogenetic analysis of these deduced proteins along with those from other insect species suggests that AgCht5-1 is orthologous to the Cht5 proteins identified in other insect species. The differences in expression patterns of these genes at different developmental stages further support that these genes may have distinct functions. Additional searching of the genomes of two other mosquito species led to the discovery of four Cht5-like genes in Aedes aegypti and three in Culex quinquefasciatus. Thus, the presence of a Cht5 gene cluster appears to be unique to mosquito species and these genes may have resulted from gene tandem duplications.     

cDNA cloning,expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi

In this study, we report a novel cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Bh-EGase II) belonging to the glycoside hydrolase family (GHF) 45 from the beetle Batocera horsfieldi. The Bh-EGase II gene spans 720 bp and consists of a single exon coding for 239 amino acid residues. Bh-EGase II showed 93.72% protein sequence identity to Ag-EGase II from the beetle Apriona germari. The GHF 45 catalytic site is conserved in Bh-EGase II. Bh-EGase II has three putative N-glycosylation sites at 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The cDNA encoding Bh-EGase II was expressed in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II from BmN cells and larval hemolymph had an enzymatic activity of approximately 928 U/mg. The enzymatic catalysis of recombinant Bh-EGase II showed the highest activity at 50 °C and pH 6.0.     

The N-terminal region of an entomopoxvirus fusolin is essential for the enhancement of peroral infection, whereas the C-terminal region is eliminated in digestive juice

The spindles of Anomala cuprea entomopoxvirus (AncuEPV), which are composed of glycoprotein fusolin, are known to enhance the peroral infectivity of AncuEPV itself and of nucleopolyhedroviruses. This has been demonstrated to involve the disruption of intestinal peritrophic membrane (PM), composed of chitin matrix, glycosaminoglycans, and proteins. To identify essential and nonessential regions for this enhancement activity, AncuEPV fusolin and its deletion mutants were expressed in Sf21 cells using a baculovirus system, and their enhancement abilities were analyzed. The recombinant fusolin enhanced the peroral infectivity of Bombyx mori nucleopolyhedrovirus up to 320-fold and facilitated the infection of host insect with AncuEPV. Deletion mutagenesis revealed that the N-terminal region (amino acids 1 to 253), a possible chitin-binding domain, is essential for the enhancement of infection, whereas the C-terminal region is entirely dispensable. The glycosylation-defective mutants N191Q, whose Asn₁₉₁ is replaced with Gln, and ΔSIG, whose signal peptide is deleted, showed considerably reduced and abolished enhancing activities, respectively, indicating that the carbohydrate chain is important in the enhancing activity. Interestingly, the C-terminal dispensable region was digested by a serine protease(s) in insect digestive juice. Moreover, both the N-terminal conserved region and the carbohydrate chain were necessary not only for chitin binding but also for stability in digestive juice. A triple amino acid replacement mutant, IHE (Ile-His-Glu₁₆₁ to Ala-Ala-Ala), was stable in digestive juice and had chitin-binding ability but did not retain its enhancing activity. These results suggest that the enhancement of infectivity involves more than the tolerance to digestive juice and chitin-binding ability.