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Pucciniales are fungal pathogens of plants that cause devastating rust diseases in agriculture. Chemically-synthesized pesticides help farmers to control rust epidemics, but governing bodies aim at limiting their use over the next decade. Defense peptides with antimicrobial activities may help to innovate a next generation of phytosanitary products for sustainable crop protection. This review comprehensively inventories the proteins or peptides exhibiting a biochemically-demonstrated antifungal activity toward Pucciniales (i.e., anti-rust proteins or peptides; hereafter ‘ARPs’), and also analyses the bioassays used to characterize them. In total, the review scrutinizes sixteen publications, which collectively report 35 ARPs. These studies used either in vitro or in planta bioassays, or a combination of both, to characterize ARPs; mostly by evaluating their ability to inhibit the spore germination process in vitro or to inhibit fungal growth and rust disease development in planta. Also, the manuscript shows that almost no mode of action against rust fungi was elucidated, although some might be inferred from studies performed on other fungi. This short review may serve as a knowledge and methodological basis to inform future studies addressing ARPs.

Abstract Background Avidins are biotin-binding proteins commonly found in the vertebrate eggs. In addition to streptavidin from Streptomyces avidinii, a growing number of avidins have been characterized from divergent bacterial species. However, a systematic research concerning their taxonomy and ecological role has never been done. We performed a search for avidin encoding genes among bacteria using available databases and classified potential avidins according to taxonomy and the ecological niches utilized by host bacteria. Results Numerous avidin-encoding genes were found in the phyla Actinobacteria and Proteobacteria. The diversity of protein sequences was high and several new variants of genes encoding biotin-binding avidins were found. The living strategies of bacteria hosting avidin encoding genes fall mainly into two categories. Human and animal pathogens were overrepresented among the found bacteria carrying avidin genes. The other widespread category were bacteria that either fix nitrogen or live in root nodules/rhizospheres of plants hosting nitrogen-fixing bacteria. Conclusions Bacterial avidins are a taxonomically and ecologically diverse group mainly found in Actinobacteria, Proteobacteria and Bacteroidetes, associated often with plant invasiveness. Avidin encoding genes in plasmids hint that avidins may be horizontally transferred. The current survey may be used as a basis in attempts to understand the ecological significance of biotin-binding capacity.

Main conclusion: A chitin-binding domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions. Complementary DNA encoding the antifungal chitinase of gazyumaru (Ficus microcarpa), designated GlxChiB, was cloned and expressed in Escherichia coli cells. The results of cDNA cloning showed that the precursor of GlxChiB has an N-terminal endoplasmic reticulum targeting signal and C-terminal vacuolar targeting signal, whereas mature GlxChiB is composed of an N-terminal carbohydrate-binding module family-18 domain (CBM18) and a C-terminal glycoside hydrolase family-19 domain (GH19) with a short linker. To clarify the role of the CBM18 domain in the antifungal activity of chitinase, the recombinant GlxChiB (wild type) and its catalytic domain (CatD) were used in quantitative antifungal assays under different ionic strengths and microscopic observations against the fungus Trichoderma viride. The antifungal activity of the wild type was stronger than that of CatD under all ionic strength conditions used in this assay; however, the antifungal activity of CatD became weaker with increasing ionic strength, whereas that of the wild type was maintained. The results at high ionic strength further verified the contribution of the CBM18 domain to the antifungal ability of GlxChiB. The microscopic observations clearly showed that the wild type acted on both the tips and the lateral wall of fungal hyphae, while CatD acted only on the tips. These results suggest that the CBM18 domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions. [ABSTRACT FROM AUTHOR]

Background: Avidins are biotin-binding proteins commonly found in the vertebrate eggs. In addition to streptavidin from Streptomyces avidinii, a growing number of avidins have been characterized from divergent bacterial species. However, a systematic research concerning their taxonomy and ecological role has never been done. We performed a search for avidin encoding genes among bacteria using available databases and classified potential avidins according to taxonomy and the ecological niches utilized by host bacteria. Results: Numerous avidin-encoding genes were found in the phyla Actinobacteria and Proteobacteria. The diversity of protein sequences was high and several new variants of genes encoding biotin-binding avidins were found. The living strategies of bacteria hosting avidin encoding genes fall mainly into two categories. Human and animal pathogens were overrepresented among the found bacteria carrying avidin genes. The other widespread category were bacteria that either fix nitrogen or live in root nodules/rhizospheres of plants hosting nitrogen-fixing bacteria. Conclusions: Bacterial avidins are a taxonomically and ecologically diverse group mainly found in Actinobacteria, Proteobacteria and Bacteroidetes, associated often with plant invasiveness. Avidin encoding genes in plasmids hint that avidins may be horizontally transferred. The current survey may be used as a basis in attempts to understand the ecological significance of biotin-binding capacity. [ABSTRACT FROM AUTHOR]

Key message: SBTX has defensive role against C. kikuchii, and therefore, its constituent genes SBTX17 and SBTX27 are promising candidates to engineer pathogen resistant plants. Soybean (Glycine max [L.] Merr.) is economically the most important legume crop in the world. Its productivity is strongly affected by fungal diseases, which reduce soybean production and seed quality and cause losses of billions of dollars worldwide. SBTX is a protein that apparently takes part in the defensive chemical arsenal of soybean against pathogens. This current study provides data that reinforce this hypothesis. Indeed, SBTX inhibited in vitro the mycelial growth of Cercospora kikuchii, it is constitutively located in the epidermal region of the soybean seed cotyledons, and it is exuded from mature imbibed seeds. Moreover, RT-qPCR analysis of the SBTX associated genes, SBTX17 and SBTX27, which encode for the 17 and 27 kDa polypeptide chains, showed that both genes are expressed in all studied plant tissues during the soybean development, with the highest levels found in the mature seeds and unifoliate leaves. In addition, to assess a local response of the soybean secondary leaves from 35-day-old plants, they were inoculated with C. kikuchii and treated with salicylic acid. It was verified using RT-qPCR that SBTX17 and SBTX27 genes overexpressed in leaves compared to controls. These findings strongly suggest that SBTX has defensive roles against C. kikuchii. Therefore, SBTX17 and SBTX27 genes are promising candidates to engineer pathogen resistant plants. [ABSTRACT FROM AUTHOR]

To establish a successful infection, a virus needs to replicate and move cell-to-cell efficiently. We investigated whether one of the genes upregulated in Nicotiana benthamiana after Bamboo mosaic virus (BaMV) inoculation was involved in regulating virus movement. We revealed the gene to be a plasma membrane-associated cation-binding protein 1-like protein, designated NbPCaP1L. The expression of NbPCaP1L in N. benthamiana was knocked down using Tobacco rattle virus-based gene silencing and consequently the accumulation of BaMV increased significantly to that of control plants. Further analysis indicated no significant difference in the accumulation of BaMV in NbPCaP1L knockdown and control protoplasts, suggesting NbPCaP1L may affect cell-to-cell movement of BaMV. Using a viral vector expressing green fluorescent protein in the knockdown plants, the mean area of viral focus, as determined by fluorescence, was found to be larger in NbPCaP1L knockdown plants. Orange fluorescence protein (OFP)-fused NbPCaP1L, NbPCaP1L-OFP, was expressed in N. benthamiana and reduced the accumulation of BaMV to 46%. To reveal the possible interaction of viral protein with NbPCaP1L, we performed yeast two-hybrid and co-immunoprecipitation experiments. The results indicated that NbPCaP1L interacted with BaMV replicase. The results also suggested that NbPCaP1L could trap the BaMV movement RNP complex via interaction with the viral replicase in the complex and so restricted viral cell-to-cell movement. [ABSTRACT FROM AUTHOR]

In this work, we analyzed the effects of coffee seed proteins, especially Cc-LTP on the larval development of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae), a bruchid pest of beans and the most important insect pest of Vigna unguiculata (L.) Walp. Artificial seed assay, which incorporated the F/0-90 fraction from Coffea canephora seeds, resulted in the reduction of oviposition and caused an inhibition of C. maculatus larval development in a dose-dependent manner. The F/0-90 fraction used at a 4 % concentration resulted in the survival of no larvae. The purified Cc-LTP, at a concentration of 0.5 %, also demonstrated effective inhibition of larval development, reducing both females oviposition and the weight and number of larvae. Cc-LTP was also able to inhibit the C. maculatus gut α-amylase activity, and immunolabeling by an anti-LTP serum was observed in the midgut tissues of the C. maculatus larvae. Cc-LTP has shown binding affinity towards microvillar cells, endoplasmic reticulum and mitochondria, as demonstrated by micrographic images taken by a transmission electron microscope. The results from this study indicate that Cc-LTP has insecticidal actions toward C. maculatus and exerts anti-nutritional effects with direct actions on intestinal tissues. [ABSTRACT FROM AUTHOR]

A 5.4-kDa antifungal peptide was purified from Phaseolus vulgaris L. cv. 'northeast red bean' using a protocol that entailed affinity chromatography, ion exchange chromatography, and gel filtration. The molecular mass was determined by matrix-assisted laser desorption ionization time-of-flight. The N-terminal amino acid sequence of the peptide was highly homologous to defensins and defensin-like peptides from several plant species. The peptide impeded the growth of a number of pathogenic fungi, including Mycosphaerella arachidicola Khokhr. (IC = 1.7 μM), Setosphaeria turcica Luttr., Fusarium oxysporum Schltdl., and Valsa mali Miyabe & G. Yamada. Antifungal activity of the peptide was fully preserved at temperatures up to 100 °C and pH values from 0 to 12. Congo red deposition at the hyphal tip of M. arachidicola was detected after exposure to the peptide, signifying that the peptide had suppressed hyphal growth. The antifungal peptide did not manifest antiproliferative activity toward human breast cancer MCF7 cells and hepatoma HepG2 cells, in contradiction to the bulk of previously reported plant defensins. The data suggest distinct structural requirements for antifungal and antiproliferative activities. [ABSTRACT FROM AUTHOR]

Glyoxal oxidase (GLOX), an extracellular HO-producing enzyme, has been reported in Phanerochaete chrysosporium and Ustilago maydis. We previously isolated a grapevine GLOX gene from the highly resistant to Erysiphe necator Chinese wild Vitis pseudoreticulata accession Baihe-35-1 and designated it as VpGLOX (GenBank accession no. DQ201181). Transient expression of VpGLOX can suppress Powdery Mildew in susceptible genotype were studied. To further investigate the function of the VpGLOX gene, real-time PCR and Western blot analysis were performed to examine expression patterns at transcriptional and translational levels, respectively. The results showed that VpGLOX expression at the transcriptional level increased significantly in the disease-resistant accession Baihe-35-1 after Erysiphe necator inoculation, but no significant changes in the susceptible accession, V. pseudoreticulata accession Guangxi-2 could be observed. As evident from a Western blot analysis, VpGLOX protein increased slightly in Baihe-35-1 after E. necator inoculation, but not statistical significant difference changes in Guangxi-2. The immunolocalization via immunogold electron microscopy showed that VpGLOX was mainly located in the adaxial epidermal cell wall of E. necator-inoculated leaves of both Baihe-35-1 and Guangxi-2. Agrobacterium-mediated transient expression assays revealed that VpGLOX expression could produce HO, which may directly play a role in defense mechanism during plant-pathogen interactions. Our results could provide further insight into the biological role of VpGLOX in the defense response against E. necator in V. pseudoreticulata. [ABSTRACT FROM AUTHOR]

Abstract: Latex and other exudates in plants contain various proteins that are thought to play important defensive roles against herbivorous insects and pathogens. Herein, the defensive effects of phloem exudates against the Eri silkworm, Samia ricini (Saturniidae, Lepidoptera) in several cucurbitaceous plants were investigated. It was found that phloem exudates are responsible for the defensive activities of cucurbitaceous plants, such as the wax gourd Benincasa hispida and Cucumis melo, especially in B. hispida, whose leaves showed the strongest growth-inhibitory activity of all the cucurbitaceous plants tested. A 35kDa proteinaceous growth-inhibitory factor against insects designated BPLP (B. hispida Phloem Lectin-like Protein) was next isolated and purified from the B. hispida exudate, using anion exchange and gel filtration chromatography. A very low concentration (70μg/g) of BPLP significantly inhibited growth of S. ricini larvae. The full-length cDNA (1076bp) encoding BPLP was cloned and its nucleotide sequence was determined. The deduced amino acid sequence of BPLP had 51% identity with a cucurbitaceous phloem lectin (phloem protein 2, PP2), and showed binding specificity to oligomers of N-acetylglucosamine. Some features of BPLP indicated that it does not have a cysteine residue and it is composed of two repeats of similar sequences, suggesting that BPLP is distinct from PP2. Recombinant BPLP, obtained by expressing the cDNA in Escherichia coli, showed both chitin-binding lectin activity and growth-inhibitory activity against S. ricini larvae. The present study thus provides experimental evidence that phloem exudates of Cucurbitaceae plants, analogous to plant latex, play defensive roles against insect herbivores, especially against chewing insects, and contain defensive substances toxic to them. [Copyright &y& Elsevier]

Among the Hawaiian papaya (Carica papaya L.) cultivars, 'Kamiya' is more tolerant to Phytophthora palmivora than 'SunUp'. To understand the molecular basis for the difference between these two cultivars, their protein profiles cultivars were investigated by two-dimensional (2D) electrophoresis. Two hundred and fifty expressed protein spots were compared between 'Kamiya' and 'SunUp' on triplicate 2D gels. Twentyfive out of 28 differentially-expressed spots were successfully identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and three differentially migrating spots were identified as a single protein. Among the 15 proteins up-regulated 2-fold or higher in 'Kamiya', nine were related to plant disease resistance and defense response, while among the 8 proteins up-regulated 2 fold or more in 'SunUp', one was involved in disease and defense. Six differentially expressed proteins were further confirmed by semi-quantitative RT-PCR. Identified proteins were involved in at least five categories of plant defense or disease resistance: jasmonic acid (JA)-dependent pathway, ATP binding cassette (ABC) transporters, plant brassinolide hormone, abscisic acid (ABA)/reactive oxygen species (ROS) plant defense pathway, and transcription regulation. Our results infer that tolerant 'Kamiya' possesses various defense mechanisms against P. palmivora. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Three plant proteinase inhibitors BbKI (kallikrein inhibitor) and BbCI (cruzipain inhibitor) from Bauhinia bauhinioides, and a BrTI (trypsin inhibitor) from B. rufa, were examined for other effects in Callosobruchus maculatus development; of these only BrTI affected bruchid emergence. BrTI and BbKI share 81% identities in their primary sequences and the major differences between them are the regions comprising the RGD and RGE motifs in BrTI. These sequences were shown to be essential for BrTI insecticidal activity, since a modified BbKI [that is a recombinant form (BbKIm) with some amino acid residues replaced by those found in BrTI sequence] also strongly inhibited insect development. By using synthetic peptides related to the BrTI sequence, YLEAPVARGDGGLA-NH2 (RGE) and IVYYPDRGETGL-NH2 (RGE), it was found that the peptide with an RGE sequence was able to block normal development of C. maculatus larvae (ED50 0.16% and LD50 0.09%), this being even more effective than the native protein. [Copyright &y& Elsevier]

Plants respond differently to damage by different herbivorous insects. We speculated that sibling herbivorous species with different host ranges might also influence plant responses differently. Such differences may be associated with the diet breadth (specialization) of herbivores within a feeding guild, and the specialist may cause less intensive plant responses than the generalist. The tobacco Nicotinana tabacum L. is the common host plant of a generalist Helicoverpa armigera (Hübner) and a specialist H. assulta Guenée (Lepidoptera, Noctuidae). The induced responses of tobacco to feeding of these two noctuid herbivores and mechanical wounding were compared. The results showed that the feeding of the specialist H. assulta and the generalist H. armigera resulted in the same inducible defensive system, but response intensity of plants was different to these two species. Inductions of jasmonic acid (JA), lipoxygenase (LOX), and proteinase inhibitors (PIs) were not significantly different concerning these two species, but H. assulta caused the less intensive foliar polyphenol oxidase (PPO) increase, more intensive nicotine and peroxidase (POD) increases in tobacco than H. armigera. The defensive response of plant to herbivores with different diet breadth seems to be more complicated than we expected, and the specialist does not necessarily cause less intensive plant responses than the generalist. [ABSTRACT FROM AUTHOR]

Abstract: Seeds of a tepary bean cultivar (Phaseolus acutifolius A. Gray) contain two isoforms of α-amylase inhibitor, designated as αAI-Pa1 and αAI-Pa2, that differ in their specificity for insect α-amylases. Like αAI-1 and αAI-2 of common bean (Phaseolus vulgaris L.), the active form of αAI-Pa2 is a heterotetramer of two α and two β subunits that are both generated from a common precursor, whereas αAI-Pa1 is composed of a single polypeptide. We have now isolated cDNAs encoding αAI-Pa1 and αAI-Pa2 and found that the deduced amino acid sequence αAI-Pa2 is most similar to that of αAI-2 whereas that of αAI-Pa1 most closely resembles those of seed lectins of tepary bean and common bean. Expression of these cDNA clones in azuki bean (Vigna angularis Willd. Ohwi & Ohashi) revealed that active forms of the inhibitor proteins accumulated in seeds and exhibited specificities for insect α-amylases identical to those of the native proteins purified from tepary bean seeds. The active form of αAI-Pa1 in transgenic azuki bean was a single polypeptide with a size similar to that expected for the full-length encoded protein. These results suggest that αAI-Pa1 defines a new type of bean αAI that is structurally related to lectins and is not activated by proteolytic processing. [Copyright &y& Elsevier]

Two distinct strategies for plant defense proteins and microbial attack proteins to target the peritrophic membrane (PM) are becoming clear. While fusolin, enhancin, Mir1-CP, chitinases, and lectins disrupt the PM to enhance the infection of microbes and toxicity of toxic proteins, and destroy the compartmentation necessary for efficient digestive processes, the MLX56 family proteins, in contrast, modify PM into thicker membrane that functions as an obstacle of nutritive processes. • PM (peritrophic membrane) is a target of microbial attack/plant defense proteins. • PM-targeting proteins function either by disrupting PM or thickening PM. • Disruption of PM by fusolin and enhancin enhances microbial infection to insects. • Disruption of PM by Mir1-CP, chitinase, and lectin relates to their defense function. • PM thickened by MLX56 inhibits insect growth as an obstacle to digestive processes. The peritrophic membrane (or peritrophic matrix: PM) is a thin membranous structure that lies along the midgut epithelium in the midgut lumen and consists of chitin and proteins. PM exists between ingested food material and midgut epithelium cells and it is on the frontline of insect-plant and insect-microbe interactions. Therefore, proteins that play major roles in plant defense against herbivorous insects and in microbial attack on insects should penetrate, destroy or modify the PM to accomplish their roles. Recently, it has become clear that some proteins crucial to plant defense or microbial attack have the PM as their primary target. In addition, several plant defense proteins have been reported to affect the PM, although it is still unclear whether the PM is their primary target. This review introduces several of these proteins: fusolin and enhancin, two proteins produced by insect viruses that greatly enhance infection of the viruses by disrupting the PM; the MLX56 family proteins found in mulberry latex as defense proteins against insect herbivores, which modify the PM to a thick structure that inhibits digestive processes; Mir1-CP, a defense cysteine protease from maize that inhibits the growth of insects at very low concentrations and degrades the PM structures; and chitinases and lectins. The importance, necessary characteristics, and modes of action of PM-targeting proteins are then discussed from a strategic point of view, by spotlighting the importance of selective permeability of the PM. Finally, the review discusses the possibility of applying PM-targeting proteins for the control of pest insects. [ABSTRACT FROM AUTHOR]

To establish a successful infection, a virus needs to replicate and move cell-to-cell efficiently. We investigated whether one of the genes upregulated in Nicotiana benthamiana after Bamboo mosaic virus (BaMV) inoculation was involved in regulating virus movement. We revealed the gene to be a plasma membrane-associated cation-binding protein 1-like protein, designated NbPCaP1L. The expression of NbPCaP1L in N. benthamiana was knocked down using Tobacco rattle virus-based gene silencing and consequently the accumulation of BaMV increased significantly to that of control plants. Further analysis indicated no significant difference in the accumulation of BaMV in NbPCaP1L knockdown and control protoplasts, suggesting NbPCaP1L may affect cell-to-cell movement of BaMV. Using a viral vector expressing green fluorescent protein in the knockdown plants, the mean area of viral focus, as determined by fluorescence, was found to be larger in NbPCaP1L knockdown plants. Orange fluorescence protein (OFP)-fused NbPCaP1L, NbPCaP1L-OFP, was expressed in N. benthamiana and reduced the accumulation of BaMV to 46%. To reveal the possible interaction of viral protein with NbPCaP1L, we performed yeast two-hybrid and co-immunoprecipitation experiments. The results indicated that NbPCaP1L interacted with BaMV replicase. The results also suggested that NbPCaP1L could trap the BaMV movement RNP complex via interaction with the viral replicase in the complex and so restricted viral cell-to-cell movement., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)