Effects of Bioinsecticidal Aegerolysin-Based Cytolytic Complexes on Non-Target Organisms.

Aegerolysin proteins ostreolysin A6 (OlyA6), pleurotolysin A2 (PlyA2) and erylysin A (EryA) produced by the mushroom genus Pleurotus bind strongly to an invertebrate-specific membrane sphingolipid, and together with a protein partner pleurotolysin B (PlyB), form transmembrane pore complexes. This pore formation is the basis for the selective insecticidal activity of aegerolysin/PlyB complexes against two economically important coleopteran pests: the Colorado potato beetle and the western corn rootworm. In this study, we evaluated the toxicities of these aegerolysin/PlyB complexes using feeding tests with two ecologically important non-target arthropod species: the woodlouse and the honey bee. The mammalian toxicity of the EryA/PlyB complex was also evaluated after intravenous administration to mice. None of the aegerolysin/PlyB complexes were toxic against woodlice, but OlyA6/PlyB and PlyA2/PlyB were toxic to honeybees, with 48 h mean lethal concentrations (LC50) of 0.22 and 0.39 mg/mL, respectively, in their food. EryA/PlyB was also tested intravenously in mice up to 3 mg/kg body mass, without showing toxicity. With no toxicity seen for EryA/PlyB for environmentally beneficial arthropods and mammals at the tested concentrations, these EryA/PlyB complexes are of particular interest for development of new bioinsecticides for control of selected coleopteran pests.

Glyceollin Transcription Factor GmMYB29A2 Regulates Soybean Resistance to Phytophthora sojae.

Glyceollin isomers I, II, and III are the major pathogen-elicited secondary metabolites (i.e. phytoalexins) of soybean (Glycine max) that, collectively with other 5-deoxyisoflavonoids, provide race-specific resistance to Phytophthora sojae. The NAC-family transcription factor (TF) GmNAC42-1 is an essential regulator of some but not all glyceollin biosynthesis genes, indicating other essential TF(s) of the glyceollin gene regulatory network remain to be identified. Here, we conducted comparative transcriptomics on soybean hairy roots of the variety Williams 82 and imbibing seeds of Harosoy 63 upon treatment with wall glucan elicitor from P. sojae and identified two homologous R2R3-type MYB TF genes, GmMYB29A1 and GmMYB29A2, up-regulated during the times of peak glyceollin biosynthesis. Overexpression and RNA interference silencing of GmMYB29A2 increased and decreased expression of GmNAC42-1, GmMYB29A1, and glyceollin biosynthesis genes and metabolites, respectively, in response to wall glucan elicitor. By contrast, overexpressing or silencing GmMYB29A1 decreased glyceollin I accumulation with marginal or no effects on the expressions of glyceollin synthesis genes, suggesting a preferential role in promoting glyceollin turnover and/or competing biosynthetic pathways. GmMYB29A2 interacted with the promoters of two glyceollin I biosynthesis genes in vitro and in vivo. Silencing GmMYB29A2 in Williams 82, a soybean variety that encodes the resistance gene Rps1k, rendered it compatible with race 1 P. sojae, whereas overexpressing GmMYB29A2 rendered the susceptible Williams variety incompatible. Compatibility and incompatibility coincided with reduced and enhanced accumulations of glyceollin I but not other 5-deoxyisoflavonoids. Thus, GmMYB29A2 is essential for accumulation of glyceollin I and expression of Phytophthora resistance.

Metabolic flux towards the (iso)flavonoid pathway in lignin modified alfalfa lines induces resistance against Fusarium oxysporum f. sp. medicaginis.

Downregulation of lignin in alfalfa (Medicago sativa L.) is associated with increased availability of cell wall polysaccharides in plant cells. We tested transgenic alfalfa plants downregulated for Caffeoyl-CoA O-methyltransferase (CCoAOMT) against an economically important fungal disease of alfalfa, Fusarium wilt caused by Fusarium oxysporum f. sp. medicaginis, and found it more resistant to this disease. Transcriptomic and metabolomic analyses indicated that the improved disease resistance against Fusarium wilt is due to increased accumulation and/or spillover of flux towards the (iso)flavonoid pathway. Some (iso)flavonoids and their pathway intermediate compounds showed strong accumulation in CCoAOMT downregulated plants after F. oxysporum f. sp. medicaginis inoculation. The identified (iso)flavonoids, including medicarpin and 7,4'-dihydroxyflavone, inhibited the in vitro growth of F. oxysporum f. sp. medicaginis. These results suggested that the increased accumulation and/or shift/spillover of flux towards the (iso)flavonoid pathway in CCoAOMT downregulated plants is associated with induced disease resistance.

Effect of lead treatment on medicarpin accumulation and on the gene expression of key enzymes involved in medicarpin biosynthesis in Medicago sativa L.

Lead (Pb) is the most common heavy metal contaminant in the environment. The present study was undertaken to determine the effect of Pb treatment on medicarpin production and accumulation in Medicago sativa L. To this aim, 7- and 30-day-old plants were treated with 0, 120, 240, 500, and 1,000 µM Pb during 10 days. The content of medicarpin was determined by HPLC, and the extent of medicarpin production was deduced from the result of semiquantitative RT-PCR performed on PAL, CHS, and VR genes. HPLC results indicated that medicarpin concentration has been reduced in the roots, while its exudation to the culture medium has been increased. RT-PCR results indicated that the transcript levels of PAL, CHS, and VR genes have not been affected following Pb stress in seedlings. At the vegetative stage, transcript levels of PAL and CHS genes have been reduced in the roots. However, the transcript level of VR gene increased at 120 and 240 µM Pb, while it decreased at higher concentrations. In the shoot, the transcript levels of PAL, CHS, and VR genes were increased following increased concentration of lead in the medium. Overall, q-PCR results suggest that medicarpin biosynthesis has been induced in the shoots and reduced in the roots of the plants treated with a toxic concentration of Pb.

Nuclease released by Verticillium dahliae is a signal for non-host resistance.

A DNase released from the fungal pathogen of bean, Fusarium solani f. sp. phaseoli (Fsph), was previously shown to signal the activation of total disease resistance and activate pathogenesis-related (PR) genes in pea. Data in the current study which used the pea-endocarp model to research non-host resistance, indicated that DNase released by Verticillium dahliae (Vd), pathogenic on potato also has non-host resistance-inducing capabilities in peas. Other strains of Vd that release DNase are pathogenic on other plant species. DNase catalytic activity was also released from representative genera of other pathogenic fungi. Purified VdDNase induced pisatin and pea gene DRR49 (PR-10 gene) in pea endocarp tissue. VdDNase reduced the in vitro growth of Vd but completely inhibited that of F. solani f. sp. pisi (Fspi) and a Colletotrichum pathogen of potato. VdDNase (2 units) applied to pea endocarp tissue both broke resistance to Fsph and increased resistance to Fspi. Pea DNA damage generated both by the VdDNase enzyme and the intact Vd spores indicated that the host DNA alteration is a component of the non-host resistance response (innate immunity). These data support the previously reported inductive potential of fungal DNase and further implicate fungal DNases as signals in activating non-host resistance responses.

Molecular cloning and characterization of a cDNA for pterocarpan 4-dimethylallyltransferase catalyzing the key prenylation step in the biosynthesis of glyceollin, a soybean phytoalexin.

Glyceollins are soybean (Glycine max) phytoalexins possessing pterocarpanoid skeletons with cyclic ether decoration originating from a C5 prenyl moiety. Enzymes involved in glyceollin biosynthesis have been thoroughly characterized during the early era of modern plant biochemistry, and many genes encoding enzymes of isoflavonoid biosynthesis have been cloned, but some genes for later biosynthetic steps are still unidentified. In particular, the prenyltransferase responsible for the addition of the dimethylallyl chain to pterocarpan has drawn a large amount of attention from many researchers due to the crucial coupling process of the polyphenol core and isoprenoid moiety. This study narrowed down the candidate genes to three soybean expressed sequence tag sequences homologous to genes encoding homogentisate phytyltransferase of the tocopherol biosynthetic pathway and identified among them a cDNA encoding dimethylallyl diphosphate: (6aS, 11aS)-3,9,6a-trihydroxypterocarpan [(-)-glycinol] 4-dimethylallyltransferase (G4DT) yielding the direct precursor of glyceollin I. The full-length cDNA encoding a protein led by a plastid targeting signal sequence was isolated from young soybean seedlings, and the catalytic function of the gene product was verified using recombinant yeast microsomes. Expression of the G4DT gene was strongly up-regulated in 5 to 24 h after elicitation of phytoalexin biosynthesis in cultured soybean cells similarly to genes associated with isoflavonoid pathway. The prenyl part of glyceollin I was demonstrated to originate from the methylerythritol pathway by a tracer experiment using [1-(13)C]Glc and nuclear magnetic resonance measurement, which coincided with the presumed plastid localization of G4DT. The first identification of a pterocarpan-specific prenyltransferase provides new insights into plant secondary metabolism and in particular those reactions involved in the disease resistance mechanism of soybean as the penultimate gene of glyceollin biosynthesis.