Metabolic responses of willow (Salix purpurea L.) leaves to mycorrhization as revealed by mass spectrometry and (1)H NMR spectroscopy metabolite profiling.

UNLABELLED: The root system of most terrestrial plants form symbiotic interfaces with arbuscular mycorrhizal fungi (AMF), which are important for nutrient cycling and ecosystem sustainability. The elucidation of the undergoing changes in plants' metabolism during symbiosis is essential for understanding nutrient acquisition and for alleviation of soil stresses caused by environmental cues. Within this context, we have undertaken the task of recording the fluctuation of willow (Salix purpurea L.) leaf metabolome in response to AMF inoculation. The development of an advanced metabolomics/bioinformatics protocol employing mass spectrometry (MS) and (1)H NMR analyzers combined with the in-house-built metabolite library for willow (http://willowmetabolib. RESEARCH: mcgill.ca/index.html) are key components of the research. Analyses revealed that AMF inoculation of willow causes up-regulation of various biosynthetic pathways, among others, those of flavonoid, isoflavonoid, phenylpropanoid, and the chlorophyll and porphyrin pathways, which have well-established roles in plant physiology and are related to resistance against environmental stresses. The recorded fluctuation in the willow leaf metabolism is very likely to provide AMF-inoculated willows with a significant advantage compared to non-inoculated ones when they are exposed to stresses such as, high levels of soil pollutants. The discovered biomarkers of willow response to AMF inoculation and corresponding pathways could be exploited in biomarker-assisted selection of willow cultivars with superior phytoremediation capacity or genetic engineering programs.

Identification of signatory secondary metabolites during mycoparasitism of Rhizoctonia solani by Stachybotrys elegans.

Stachybotrys elegans is able to parasitize the fungal plant pathogen Rhizoctonia solani AG-3 following a complex and intimate interaction, which, among others, includes the production of cell wall-degrading enzymes, intracellular colonization, and expression of pathogenic process encoding genes. However, information on the metabolome level is non-existent during mycoparasitism. Here, we performed a direct-infusion mass spectrometry (DIMS) metabolomics analysis using an LTQ Orbitrap analyzer in order to detect changes in the profiles of induced secondary metabolites of both partners during this mycoparasitic interaction 4 and 5 days following its establishment. The diketopiperazine(s) (DKPs) cyclo(S-Pro-S-Leu)/cyclo(S-Pro-S-Ile), ethyl 2-phenylacetate, and 3-nitro-4-hydroxybenzoic acid were detected as the primary response of Rhizoctonia 4 days following dual-culturing with Stachybotrys, whereas only the latter metabolite was up-regulated 1 day later. On the other hand, trichothecenes and atranones were mycoparasite-derived metabolites identified during mycoparasitism 4 and 5 days following dual-culturing. All the above secondary metabolites are known to exhibit bioactivity, including fungitoxicity, and represent key elements that determine the outcome of the interaction being studied. Results could be further exploited in programs for the evaluation of the bioactivity of these metabolites per se or their chemical analogs, and/or genetic engineering programs to obtain more efficient mycoparasite strains with improved efficacy and toxicological profiles.

Suppression subtractive hybridization and comparative expression of a pore-forming toxin and glycosyl hydrolase genes in Rhizoctonia solani during potato sprout infection.

Rhizoctonia solani is a plant pathogenic fungus that causes black scurf on tubers and stem and stolon canker on underground parts of potato plant. Early in the season, the fungus attacks germinating sprouts underground before they emerge from the soil. Damage at this stage results in delayed emergence of weakened plants with poor and uneven stands. The mechanism underlying this phenomenon has been investigated in this study by coupling a cDNA-suppression subtractive hybridization (SSH) library to differential screening to identify transcripts of R. solani that are down-regulated during infection of potato sprouts. We report on the identification of 33 unique genes with functions related to carbohydrate binding, vitamin synthesis, pathogenicity, translation, ATP and nucleic acid binding and other categories. RACE-PCR was used to clone and characterize the first full-length cDNA clones, RSENDO1 and RSGLYC1 that encode for an eukaryotic delta-endotoxin CytB protein and an intracellular glycosyl hydrolase, respectively. Quantitative real-time PCR revealed the down-regulation of RSENDO1 during infection of potato sprouts and the up-regulation of RSGLYC1 when the fungus was grown on a cellulose-based nutrient medium. In contrast, additional experiments have highlighted the down-regulation of RSENDO1 when R. solani was co-cultured with the mycoparasite Stachybotrys elegans and the bacterial antagonist Bacillus subtilis B26. These results advance our understanding of R. solani-potato interaction in subterranean parts of the plant. Such approaches could be considered in building an efficient integrated potato disease management program.

Genes of the de novo and Salvage Biosynthesis Pathways of Vitamin B6 are Regulated under Oxidative Stress in the Plant Pathogen Rhizoctonia solani.

Vitamin B6 is recognized as an important cofactor required for numerous metabolic enzymes, and has been shown to act as an antioxidant and play a role in stress responses. It can be synthesized through two different routes: salvage and de novo pathways. However, little is known about the possible function of the vitamin B6 pathways in the fungal plant pathogen Rhizoctonia solani. Using genome walking, the de novo biosynthetic pathway genes; RsolPDX1 and RsolPDX2 and the salvage biosynthetic pathway gene, RsolPLR were sequenced. The predicted amino acid sequences of the three genes had high degrees of similarity to other fungal PDX1, PDX2, and PLR proteins and are closely related to other R. solani anastomosis groups. We also examined their regulation when subjected to reactive oxygen species (ROS) stress inducers, the superoxide generator paraquat, or H2O2, and compared it to the well-known antioxidant genes, catalase and glutathione-S-transferase (GST). The genes were differentially regulated with transcript levels as high as 33 fold depending on the gene and type of stress reflecting differences in the type of damage induced by ROS. Exogenous addition of the vitamers PN or PLP in culture medium significantly induced the transcription of the vitamin B6 de novo encoding genes as early as 0.5 hour post treatment (HPT). On the other hand, transcription of RsolPLR was vitamer-specific; a down regulation upon supplementation of PN and upregulation with PLP. Our results suggest that accumulation of ROS in R. solani mycelia is linked to transcriptional regulation of the three genes and implicate the vitamin B6 biosynthesis machinery in R. solani, similar to catalases and GST, as an antioxidant stress protector against oxidative stress.

Characterization and transcriptional regulation of Stachybotrys elegans mitogen-activated-protein kinase gene smkA following mycoparasitism and starvation conditions.

Mitogen-activated protein kinase (MAPK) signaling pathways play an important role in the development and conidiation of fungal pathogens on their hosts and the sensing of host-derived cues. Mycoparasitism is a fungus-fungus interaction comprising host-pathogen cross talk. Until now, only little information is available on the role of the MAPK signaling pathway during this interaction. Here, we report on the differential expression of a MAPK/ERK gene in the mycoparasite Stachybotrys elegans in response to direct parasitism of different vegetative structures of the plant pathogen Rhizoctonia solani (i.e., carbon-rich condition) and to nutrient starvation (i.e., carbon-poor condition). Western blot analysis against ERK1/2 highlighted an increase in their phosphorylated forms when S. elegans was grown under starvation condition compared to that detected in response to mycoparasitism. A higher abundance of phosphorylated ERK1/2 at the third day of interaction compared to that estimated under starvation condition was detected applying LC-MS/MS. At the transcriptional level, smkA, a YERK1 class member, was significantly induced in response to hyphal parasitism compared to parasitized sclerotia at 3, 4, and 5 days of interaction. However, under starvation condition, smkA levels were significantly induced after 7 days of growth. Southern blot analysis revealed that smkA is member of a small gene family. Collectively, these results suggest that smkA could be implicated in the mycoparasitic process in S. elegans as well as in stress-activated pathways. These results may be of wider significance in other fungus-fungus interactions.

Effects of high-temperature stress on soybean isoflavone concentration and expression of key genes involved in isoflavone synthesis.

Isoflavones have been reported to have putative health-beneficial properties, which has led to increased interest and demand for soybeans and soy-based products. This study was conducted to determine the effects of high-temperature stress on isoflavone concentration and expression of four key genes involved in isoflavone synthesis (i.e., CHS7, CHS8, IFS1, and IFS2) in both soybean pods and seeds during their late reproductive stage (i.e., R5-R8). Isoflavone concentrations were quantified using high-performance liquid chromatography (HPLC), and gene expression was studied using quantitative real-time (qRT)-PCR. High-temperature stress [33/25 °C (day/night temperatures)] imposed at the late reproductive stage (R5-R8) reduced total isoflavone concentration by 46-86 and 20-73% in seeds and pods, respectively, the reduction depending on the stage of maturity. At stage R5, the reduction in total isoflavone concentration was greater in seeds than in pods, whereas at subsequent stages, the reverse was observed. High-temperature stress had a large impact on the expression of CHS7, CHS8, IFS1, and IFS2 in both seeds and pods. In seeds, temperature stress reduced the expression of one gene at the R5 stage (CHS8), two genes at the R6 stage (CHS7 and IFS1), and all four genes at the R7 stage, the reduction ranging between 35 and 97%. In pods, high-temperature stress affected the expression of two genes at the R6 stage (CHS7 and IFS2) and all four genes at the R7 stage. Unlike in seeds, at the R6 stage, high temperature increased the expression of CHS7 and IFS2 by 72 and 736%, respectively, whereas at R7 stage the expression of all genes was reduced by an average of 97%. The present study reveals that high-temperature stress initiated at the R5 stage and maintained until maturation (i.e., R8 stage) has a rapid and sustained negative effect on isoflavone concentration in both seeds and pods. High temperature also affects gene expression; however, there was no clear correlation between isoflavone concentration and gene expression.

Expression of genes of Rhizoctonia solani and the biocontrol Stachybotrys elegans during mycoparasitism of hyphae and sclerotia.

Knowledge of mycoparasitism has been focused on how antagonists affect pathogens in relation to mechanisms, metabolites and gene expression. Just as microbial antagonists use a diverse arsenal of mechanisms to dominate interactions with hosts, hosts also have diverse responses to counteract antagonism. In this study differential gene expression of eight mycoparasitism-induced genes and eight host-response genes was monitored during in vivo interactions between the mycoparasite Stachybotrys elegans and hyphae and sclerotia of the host, Rhizoctonia solani over 5 d of interaction. Using real time reverse transcription polymerase chain reaction, comparative analyses demonstrated that hyphal and sclerotial structures triggered different expression patterns. These results indicated that multiple regulatory mechanisms might be involved. The high elevated expression of some genes belonging to the mycoparasite and the host suggest that these genes play an important role during the mycoparasitic process and host defense respectively.

AFLP reveals structural details of genetic diversity within cultivated olive germplasm from the Eastern Mediterranean.

Amplified fragment length polymorphism (AFLP) analysis was used to assess genetic inter-relationships among olive varieties cultivated in the Eastern Mediterranean Basin. The genotypes sampled included most of the important cultivars from Turkey, Greece and the Middle East and selected genotypes from the Western Mediterranean area. A total of 119 polymorphic markers were generated from five selective primer-pair combinations. The combined data sets generated by just two primer-pairs were adequate to discriminate between all 65 genotypes, while each primer-pair could individually identify up to 64 genotypes. A factorial correspondence analysis (FCA) plot indicated that the cultivars clustered into two relatively modestly defined groups. The first broad group was dominated by cultivars from Turkey but also included genotypes originating from the Middle East (Syria and Lebanon) that collectively formed a tight subcluster. The second group comprised Greek cultivars and those originating from the Western Mediterranean. A significant genetic distance value between Greek and Turkish cultivars was provided by an analysis of molecular variance (AMOVA). There was also evidence of substructure here, with an apparent separation of most Spanish and Italian clones. These findings are in general accordance to previous suggestions of an East-West divergence of olive cultivars, although the dichotomy is less extensive than reported previously and complicated by regional variation within each group.