Yariv Brotman

Role of swollenin, an expansin-like protein from Trichoderma, in plant root colonization.

Swollenin, a protein first characterized in the saprophytic fungus Trichoderma reesei, contains an N-terminal carbohydrate-binding module family 1 domain (CBD) with cellulose-binding function and a C-terminal expansin-like domain. This protein was identified by liquid chromatography-mass spectrometry among many other cellulolytic proteins secreted in the coculture hydroponics medium of cucumber (Cucumis sativus) seedlings and Trichoderma asperellum, a well-known biocontrol agent and inducer of plant defense responses. The swollenin gene was isolated and its coding region was overexpressed in the same strain under the control of the constitutive pki1 promoter. Trichoderma transformants showed a remarkably increased ability to colonize cucumber roots within 6 h after inoculation. On the other hand, overexpressors of a truncated swollenin sequence bearing a 36-amino acid deletion of the CBD did not differ from the wild type, showing in vivo that this domain is necessary for full protein activity. Root colonization rates were reduced in transformants silenced in swollenin gene expression. A synthetic 36-mer swollenin CBD peptide was shown to be capable of stimulating local defense responses in cucumber roots and leaves and to afford local protection toward Botrytis cinerea and Pseudomonas syringae pv lachrymans infection. This indicates that the CBD domain might be recognized by the plant as a microbe-associated molecular pattern in the Trichoderma-plant interaction.

Inhibition of aconitase by nitric oxide leads to induction of the alternative oxidase and to a shift of metabolism towards biosynthesis of amino acids

Nitric oxide (NO) is a free radical molecule involved in signalling and in hypoxic metabolism. This work used the nitrate reductase double mutant of Arabidopsis thaliana (nia) and studied metabolic profiles, aconitase activity, and alternative oxidase (AOX) capacity and expression under normoxia and hypoxia (1% oxygen) in wild-type and nia plants. The roots of nia plants accumulated very little NO as compared to wild-type plants which exhibited ∼20-fold increase in NO emission under low oxygen conditions. These data suggest that nitrate reductase is involved in NO production either directly or by supplying nitrite to other sites of NO production (e.g. mitochondria). Various studies revealed that NO can induce AOX in mitochondria, but the mechanism has not been established yet. This study demonstrates that the NO produced in roots of wild-type plants inhibits aconitase which in turn leads to a marked increase in citrate levels. The accumulating citrate enhances AOX capacity, expression, and protein abundance. In contrast to wild-type plants, the nia double mutant failed to show AOX induction. The overall induction of AOX in wild-type roots correlated with accumulation of glycine, serine, leucine, lysine, and other amino acids. The findings show that NO inhibits aconitase under hypoxia which results in accumulation of citrate, the latter in turn inducing AOX and causing a shift of metabolism towards amino acid biosynthesis.

Transcript and metabolite analysis of the Trichoderma-induced systemic resistance response to Pseudomonas syringae in Arabidopsis thaliana

In the present study we have assessed, by transcriptional and metabolic profiling, the systemic defence response of Arabidopsis thaliana plants to the leaf pathogen Pseudomonas syringae pv. tomato DC3000 (Pst) induced by the beneficial fungus Trichoderma asperelloides T203. Expression analysis (qPCR) of a set of 137 Arabidopsis genes related to Pst defence responses showed that T203 root colonization is not associated with major detectable transcriptomic changes in leaves. However, plants challenged with the bacterial pathogen showed quantitative differences in gene expression when pre-inoculated with T203, supporting priming of the plant by this beneficial fungus. Among the defence-related genes affected by T203, lipid transfer protein (LTP)4, which encodes a member of the lipid transfer pathogenesis-related family, is upregulated, whereas the WRKY40 transcription factor, known to contribute to Arabidopsis susceptibility to bacterial infection, shows reduced expression. On the other hand, root colonization by this beneficial fungus substantially alters the plant metabolic profile, including significant changes in amino acids, polyamines, sugars and citric acid cycle intermediates. This may in part reflect an increased energy supply required for the activation of plant defences and growth promotion effects mediated by Trichoderma species.

Inhibition of Fungal and Bacterial Plant Pathogens In Vitro and In Planta with Ultrashort Cationic Lipopeptides

ABSTRACT Plant diseases constitute an emerging threat to global food security. Many of the currently available antimicrobial agents for agriculture are highly toxic and nonbiodegradable and cause extended environmental pollution. Moreover, an increasing number of phytopathogens develop resistance to them. Recently, we have reported on a new family of ultrashort antimicrobial lipopeptides which are composed of only four amino acids linked to fatty acids (A. Makovitzki, D. Avrahami, and Y. Shai, Proc. Natl. Acad. Sci. USA 103:15997-16002, 2006). Here, we investigated the activities in vitro and in planta and the modes of action of these short lipopeptides against plant-pathogenic bacteria and fungi. They act rapidly, at low micromolar concentrations, on the membranes of the microorganisms via a lytic mechanism. In vitro microscopic analysis revealed wide-scale damage to the microorganisms membrane, in addition to inhibition of pathogen growth. In planta potent antifungal activity was demonstrated on cucumber fruits and leaves infected with the pathogen Botrytis cinerea as well as on corn leaves infected with Cochliobolus heterostrophus. Similarly, treatment with the lipopeptides of Arabidopsis leaves infected with the bacterial leaf pathogen Pseudomonas syringae efficiently and rapidly reduced the number of bacteria. Importantly, in contrast to what occurred with many native lipopeptides, no toxicity was observed on the plant tissues. These data suggest that the ultrashort lipopeptides could serve as native-like antimicrobial agents economically feasible for use in plant protection.

The form of nitrogen nutrition affects resistance against Pseudomonas syringae pv. phaseolicola in tobacco

Different forms of nitrogen (N) fertilizer affect disease development; however, this study investigated the effects of N forms on the hypersensitivity response (HR)—a pathogen-elicited cell death linked to resistance. HR-eliciting Pseudomonas syringae pv. phaseolicola was infiltrated into leaves of tobacco fed with either or . The speed of cell death was faster in -fed compared with -fed plants, which correlated, respectively, with increased and decreased resistance. Nitric oxide (NO) can be generated by nitrate reductase (NR) to influence the formation of the HR. NO generation was reduced in -fed plants where N assimilation bypassed the NR step. This was similar to that elicited by the disease-forming P. syringae pv. tabaci strain, further suggesting that resistance was compromised with feeding. PR1a is a biomarker for the defence signal salicylic acid (SA), and expression was reduced in -fed compared with fed plants at 24h after inoculation. This pattern correlated with actual SA measurements. Conversely, total amino acid, cytosolic and apoplastic glucose/fructose and sucrose were elevated in - treated plants. Gas chromatography/mass spectroscopy was used to characterize metabolic events following different N treatments. Following nutrition, polyamine biosynthesis was predominant, whilst after nutrition, flux appeared to be shifted towards the production of 4-aminobutyric acid. The mechanisms whereby feeding enhances SA, NO, and polyamine-mediated HR-linked defence whilst these are compromised with , which also increases the availability of nutrients to pathogens, are discussed.

Stress Responses to Tomato Yellow Leaf Curl Virus (TYLCV) Infection of Resistant and Susceptible Tomato Plants are Different

Two genetically close inbred tomato lines, one resistant to Tomato yellow leaf curl virus (TYLCV) infection (R), the other susceptible (S), showed completely different stress response upon TYLCV infection. S plants were stunted and do not yield, while R plants remained symptomless and yielded. Comparison of protein profiles and metabolites patterns in TYLCV infected R and S tomatoes revealed a completely different host stress response. S plants were characterized by higher levels of reactive oxygen species (ROS) and ROS compounds, the anti-oxidative, pathogenesis-related (PR) and wound-induced proteins were predominant. In contrast, infection of R tomatoes did not drastically activate the same host defense mechanisms as in S plants, while R homeostasis was much more effectively maintained by protein and chemical chaperones. Sources of carbon and nitrogen were less affected by TYLCV in R than in S plants, which could make R plants more balanced and more fit to sustain infection. Even though both tomato types contained comparable amounts of TYLCV at the specified stage of infection, the cellular immune responses were different. Presented results are preliminary and indicate not so much concrete data but the global tender in understanding of the cellular response to virus stress at the background of resistance and susceptibility to TYLCV.

Linking gene expression and membrane lipid composition of Arabidopsis.

This work examines the connection between changes in gene expression and changes in membrane lipid composition in Arabidopsis exposed to different light and temperature conditions. Three transcriptional programs identified are shown to be coordinated with changes in specific membrane lipids, further supported by lipidomic analysis of selected knockout lines. Glycerolipid metabolism of plants responds dynamically to changes in light intensity and temperature, leading to the modification of membrane lipid composition to ensure optimal biochemical and physical properties in the new environment. Although multiple posttranscriptional regulatory mechanisms have been reported to be involved in the process, the contribution of transcriptional regulation remains largely unknown. Here, we present an integrative analysis of transcriptomic and lipidomic data, revealing large-scale coordination between gene expression and changes in glycerolipid levels during the Arabidopsis thaliana response to light and temperature stimuli. Using a multivariate regression technique called O2PLS, we show that the gene expression response is strictly coordinated at the biochemical pathway level and occurs in parallel with changes of specific glycerolipid pools. Five interesting candidate genes were chosen for further analysis from a larger set of candidates identified based on their close association with various groups of glycerolipids. Lipidomic analysis of knockout mutant lines of these five genes showed a significant relationship between the coordination of transcripts and glycerolipid levels in a changing environment and the effects of single gene perturbations.

Synthetic ultrashort cationic lipopeptides induce systemic plant defense responses against bacterial and fungal pathogens.

ABSTRACT A new family of synthetic, membrane-active, ultrashort lipopeptides composed of only four amino acids linked to fatty acids was tested for the ability to induce systemic resistance and defense responses in plants. We found that two peptides wherein the third residue is a d-enantiomer (italic), C16-KKKK and C16-KLLK, can induce medium alkalinization of tobacco suspension-cultured cells and expression of defense-related genes in cucumber and Arabidopsis seedlings. Moreover, these compounds can prime systemic induction of antimicrobial compounds in cucumber leaves similarly to the plant-beneficial fungus Trichoderma asperellum T203 and provide systemic protection against the phytopathogens Botrytis cinerea B05, Pseudomonas syringae pv. lachrimans, and P. syringae pv. tomato DC3000. Thus, short cationic lipopeptides are a new category of compounds with potentially high utility in the induction of systemic resistance in plants.

Genomics-based strategies for the use of natural variation in the improvement of crop metabolism.

Next-generation genomics holds great potential in the study of plant phenotypic variation. With several crop reference genomes now available, the affordable costs of de novo genome assembly or target resequencing offer the opportunity to mine the enormous amount of genetic diversity hidden in crop wild relatives. Wide introgressions from these wild ancestors species or land races represent a possible strategy to improve cultivated varieties. In this review, we discuss the mechanisms underlying metabolic diversity within plant species and the possible strategies (and barriers) to introgress novel metabolic traits into cultivated varieties. We show how deep genomic surveys uncover various types of structural variants from extended gene pools of major crops and highlight how this variation may be used for the improvement of crop metabolism.

The LysM Receptor-Like Kinase LysM RLK1 Is Required to Activate Defense and Abiotic-Stress Responses Induced by Overexpression of Fungal Chitinases in Arabidopsis Plants

Application of crab shell chitin or pentamer chitin oligosaccharide to Arabidopsis seedlings increased tolerance to salinity in wild-type but not in knockout mutants of the LysM Receptor-Like Kinase1 (CERK1/LysM RLK1) gene, known to play a critical role in signaling defense responses induced by exogenous chitin. Arabidopsis plants overexpressing the endochitinase chit36 and hexoaminidase excy1 genes from the fungus Trichoderma asperelleoides T203 showed increased tolerance to salinity, heavy-metal stresses, and Botrytis cinerea infection. Resistant lines, overexpressing fungal chitinases at different levels, were outcrossed to lysm rlk1 mutants. Independent homozygous hybrids lost resistance to biotic and abiotic stresses, despite enhanced chitinase activity. Expression analysis of 270 stress-related genes, including those induced by reactive oxygen species (ROS) and chitin, revealed constant up-regulation (at least twofold) of 10 genes in the chitinase-overexpressing line and an additional 76 salt-induced genes whose expression was not elevated in the lysm rlk1 knockout mutant or the hybrids harboring the mutation. These findings elucidate that chitin-induced signaling mediated by LysM RLK1 receptor is not limited to biotic stress response but also encompasses abiotic-stress signaling and can be conveyed by ectopic expression of chitinases in plants.