Modes of action and potential as a peptide-based biofungicide of a plant defensin MtDef4.

Due to rapidly emerging resistance to single-site fungicides in fungal pathogens of plants, there is a burgeoning need for safe and multisite fungicides. Plant antifungal peptides with multisite modes of action (MoA) have potential as bioinspired fungicides. Medicago truncatula defensin MtDef4 was previously reported to exhibit potent antifungal activity against fungal pathogens. Its MoA involves plasma membrane disruption and binding to intracellular targets. However, specific biochemical processes inhibited by this defensin and causing cell death have not been determined. Here, we show that MtDef4 exhibited potent antifungal activity against Botrytis cinerea. It induced severe plasma membrane and organelle irregularities in the germlings of this pathogen. It bound to fungal ribosomes and inhibited protein translation in vitro. A MtDef4 variant lacking antifungal activity exhibited greatly reduced protein translation inhibitory activity. A cation-tolerant MtDef4 variant was generated that bound to ß-glucan of the fungal cell wall with higher affinity than MtDef4. It also conferred a greater reduction in the grey mould disease symptoms than MtDef4 when applied exogenously on Nicotiana benthamiana plants, tomato fruits and rose petals. Our findings revealed inhibition of protein synthesis as a likely target of MtDef4 and the potential of its cation-tolerant variant as a peptide-based fungicide.

Enhancing Crop Resilience to Drought Stress through CRISPR-Cas9 Genome Editing.

With increasing frequency and severity of droughts in various parts of the world, agricultural productivity may suffer major setbacks. Among all the abiotic factors, drought is likely to have one of the most detrimental effects on soil organisms and plants. Drought is a major problem for crops because it limits the availability of water, and consequently nutrients which are crucial for plant growth and survival. This results in reduced crop yields, stunted growth, and even plant death, according to the severity and duration of the drought, the plant's developmental stage, and the plant's genetic background. The ability to withstand drought is a highly complex characteristic that is controlled by multiple genes, making it one of the most challenging attributes to study, classify, and improve. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technology has opened a new frontier in crop enhancement, revolutionizing plant molecular breeding. The current review provides a general understanding of principles as well as optimization of CRISPR system, and presents applications on genetic enhancement of crops, specifically in terms of drought resistance and yield. Moreover, we discuss how innovative genome editing techniques can aid in the identification and modification of genes conferring drought tolerance.

Maize Antifungal Protein AFP1 Elevates Fungal Chitin Levels by Targeting Chitin Deacetylases and Other Glycoproteins.

Pathogenic fungi convert chitin to chitosan to evade plant perception and disarm chitin-triggered immune responses. Whether plants have evolved factors to counteract this evasion mechanism remains obscure. Here, we decipher the mechanism underlying the antifungal activity of maize secretory mannose-binding cysteine-rich receptor-like secreted protein (CRRSP), antifungal protein 1 (AFP1). AFP1 binds to multiple sites on the surface of sporidial cells, filaments, and germinated spores of the biotrophic fungus Ustilago maydis. It inhibits cell growth and budding, as well as spore germination. AFP1 promiscuously interacts with most chitin deacetylases (CDAs) by recognizing the conserved NodB domain to interfere with the enzyme activity. Deletion of O-mannosyltransferase 4 decreases protein mannosylation, which correlates with reduced AFP1 binding and antifungal activity, suggesting that AFP1 interacts with mannosylated proteins to exhibit an inhibitory effect. AFP1 also has extended inhibitory activity against Saccharomyces cerevisiae; however, AFP1 did not reduce binding to the double ΔΔcda1,2 mutant, suggesting the targets of AFP1 have expanded to other cell surface glycoproteins, probably facilitated by its mannose-binding property. Increasing chitin levels by modulating the activity of cell surface glycoproteins is a universal feature of AFP1 interacting with a broad spectrum of fungi to inhibit their growth. IMPORTANCE Plants alert immune systems by recognizing the fungal pathogen cell wall component chitin via pattern recognition cell surface receptors. Successful fungal pathogens escape the perception by deacetylating chitin to chitosan, which is also necessary for fungal cell development and virulence. Targeting glycoproteins that are associated with regulating chitin metabolism and maintaining cell wall morphogenesis presents an effective strategy to combat fungal pathogens by simultaneously altering cell wall plasticity, activating chitin-triggered immunity, and impairing fungal viability. Our study provides molecular insights into a plant DUF26 domain-containing secretory protein in warding off a broad range of fungal pathogens by acting on more than one glycoprotein target.

Xylanase Inhibitors: Defense Players in Plant Immunity with Implications in Agro-Industrial Processing.

Xylanase inhibitors (XIs) are plant cell wall proteins largely distributed in monocots that inhibit the hemicellulose degrading activity of microbial xylanases. XIs have been classified into three classes with different structures and inhibition specificities, namely Triticum aestivum xylanase inhibitors (TAXI), xylanase inhibitor proteins (XIP), and thaumatin-like xylanase inhibitors (TLXI). Their involvement in plant defense has been established by several reports. Additionally, these inhibitors have considerable economic relevance because they interfere with the activity of xylanases applied in several agro-industrial processes. Previous reviews highlighted the structural and biochemical properties of XIs and hypothesized their role in plant defense. Here, we aimed to update the information on the genomic organization of XI encoding genes, the inhibition properties of XIs against microbial xylanases, and the structural properties of xylanase-XI interaction. We also deepened the knowledge of XI regulation mechanisms in planta and their involvement in plant defense. Finally, we reported the recently studied strategies to reduce the negative impact of XIs in agro-industrial processes and mentioned their allergenicity potential.

Reduction of Allergenic Potential in Bread Wheat RNAi Transgenic Lines Silenced for CM3, CM16 and 0.28 ATI Genes.

Although wheat is used worldwide as a staple food, it can give rise to adverse reactions, for which the triggering factors have not been identified yet. These reactions can be caused mainly by kernel proteins, both gluten and non-gluten proteins. Among these latter proteins, α-amylase/trypsin inhibitors (ATI) are involved in baker's asthma and realistically in Non Celiac Wheat Sensitivity (NCWS). In this paper, we report characterization of three transgenic lines obtained from the bread wheat cultivar Bobwhite silenced by RNAi in the three ATI genes CM3, CM16 and 0.28. We have obtained transgenic lines showing an effective decrease in the activity of target genes that, although showing a higher trypsin inhibition as a pleiotropic effect, generate a lower reaction when tested with sera of patients allergic to wheat, accounting for the important role of the three target proteins in wheat allergies. Finally, these lines show unintended differences in high molecular weight glutenin subunits (HMW-GS) accumulation, involved in technological performances, but do not show differences in terms of yield. The development of new genotypes accumulating a lower amount of proteins potentially or effectively involved in allergies to wheat and NCWS, not only offers the possibility to use them as a basis for the production of varieties with a lower impact on adverse reaction, but also to test if these proteins are actually implicated in those pathologies for which the triggering factor has not been established yet.

Biosynthesis and tissue-specific partitioning of camphor and eugenol in Ocimum kilimandscharicum.

In Ocimum kilimandscharicum, the relative volatile composition of camphor in leaves was as high as 55%, while that of eugenol in roots was 57%. These metabolites were differentially partitioned between the aerial and root tissues. Global metabolomics revealed tissue-specific biochemical specialization, evident by the differential distribution of 2588 putative metabolites across nine tissues. Next-generation sequencing analysis indicated differential expression of 51 phenylpropanoid and 55 terpenoid pathway genes in aerial and root tissues. By integrating metabolomics with transcriptomics, the camphor biosynthesis pathway in O. kilimandscharicum was elucidated. In planta bioassays revealed the role of geranyl diphosphate synthase (gpps) and borneol dehydrogenase (bdh) in camphor biosynthesis. Further, the partitioning of camphor was attributed to tissue-specific gene expression of both the pathway entry point (gpps) and terminal (bdh) enzyme. Unlike camphor, eugenol accumulated more in roots; however, absence of the eugenol synthase gene in roots indicated long distance transport from aerial tissues. In silico co-expression analysis indicated the potential involvement of ATP-binding cassette, multidrug and toxic compound extrusion, and sugar transporters in eugenol transport. Similar partitioning was evident across five other Ocimum species. Overall, our work indicates that metabolite partitioning maybe a finely regulated process, which may have implications on plant growth, development, and defense.

Wheat ATI CM3, CM16 and 0.28 Allergens Produced in Pichia Pastoris Display a Different Eliciting Potential in Food Allergy to Wheat ‡.

Although wheat is a staple food for most of the human population, some of its components trigger adverse reactions. Among wheat components, the alpha-amylase/trypsin inhibitors (ATI) are important triggers of several allergies and activators of innate immunity. ATI are a group of exogenous protease inhibitors and include several polypeptides. The three ATI polypeptides named CM3, CM16 and 0.28 are considered major allergens, and might also play a role in other common wheat-related pathologies, such as Non Celiac Wheat Sensitivity and even Celiac Disease. On this basis, we pointed to obtain high amounts of them in purity and to evaluate their allergenicity potential. We thus isolated the mRNA corresponding to the three ATI genes CM3, CM16 and 0.28 from 28 days post-anthesis wheat kernels and the corresponding cDNAs were used for heterologous expression in Pichia pastoris. The three purified proteins were tested in degranulation assay against human sera of patients with food allergy to wheat. A large range of degranulation values was observed for each protein according to the sera tested. All of the three purified proteins CM3, CM16 and 0.28 were active as allergens because they were able to induce basophils degranulation on wheat allergic patients' sera, with the highest values of ß-hexosaminidase release observed for CM3 protein.

Structural, functional and evolutionary diversity of 4-coumarate-CoA ligase in plants.

MAIN CONCLUSIONS: The 4-coumarate-CoA ligases (4CL) contribute in channelizing flux of different phenylpropanoid biosynthetic pathways. Expression of 4CL is optimized at developmental stages and in response to environmental triggers such as biotic and abiotic stresses. The enzyme is valuable in metabolic pathway engineering for curcuminoids, resveratrol, biofuel production and nutritional improvement. Vigorous analysis of regulation at functional and expression level is obligatory to attain efficient commercial production of candidate metabolites using 4CL. Phenylpropanoid pathway provides precursors for numerous secondary metabolites in plants. In this pathway, 4-coumarate-CoA ligase (EC 6.2.1.12, 4CL) is the main branch point enzyme which generates activated thioesters. Being the last enzyme of three shared common steps in general phenylpropanoid pathway, it contributes to channelize precursors for different phenylpropanoids. In plants, 4CL enzymes are present in multiple isoforms and encoded by small gene family. It belongs to adenylate-forming enzyme family and catalyzes the reaction that converts hydroxy or methoxy cinnamic acid derivatives to corresponding thioesters. These thioesters are further utilized for biosynthesis of phenylpropanoids, which are known for having numerous nutritional and medicinal applications. In addition, the 4CL enzymes have been characterized from various plants for their role in plant physiology or in biotic and abiotic stresses. Furthermore, specific isoforms are differentially regulated upon exposure to diverse stimuli leading to flux diversion toward the particular metabolite biosynthesis. Evolutionary studies showed that 4CL separately evolved after monocot and dicot segregation. Here, we provide a comprehensive review on 4CL, which includes evolution, function, gene/protein structure, role in metabolite biosynthesis and cellular partition, and their regulation. Based on the available data, we have explored the scope for pathway engineering by utilizing 4CL enzymes.

Comprehensive metabolic and transcriptomic profiling of various tissues provide insights for saponin biosynthesis in the medicinally important Asparagus racemosus.

Asparagus racemosus (Shatavari), belongs to the family Asparagaceae and is known as a "curer of hundred diseases" since ancient time. This plant has been exploited as a food supplement to enhance immune system and regarded as a highly valued medicinal plant in Ayurvedic medicine system for the treatment of various ailments such as gastric ulcers, dyspepsia, cardiovascular diseases, neurodegenerative diseases, cancer, as a galactogogue and against several other diseases. In depth metabolic fingerprinting of various parts of the plant led to the identification of 13 monoterpenoids exclusively present in roots. LC-MS profiling led to the identification of a significant number of steroidal saponins (33). However, we have also identified 16 triterpene saponins for the first time in A. racemosus. In order to understand the molecular basis of biosynthesis of major components, transcriptome sequencing from three different tissues (root, leaf and fruit) was carried out. Functional annotation of A. racemosus transcriptome resulted in the identification of 153 transcripts involved in steroidal saponin biosynthesis, 45 transcripts in triterpene saponin biosynthesis, 44 transcripts in monoterpenoid biosynthesis and 79 transcripts in flavonoid biosynthesis. These findings will pave the way for better understanding of the molecular basis of steroidal saponin, triterpene saponin, monoterpenoids and flavonoid biosynthesis in A. racemosus.

Pyramiding PvPGIP2 and TAXI-III But Not PvPGIP2 and PMEI Enhances Resistance Against Fusarium graminearum.

Plant protein inhibitors counteract the activity of cell wall-degrading enzymes (CWDEs) secreted by pathogens to breach the plant cell-wall barrier. Transgenic plants expressing a single protein inhibitor restrict pathogen infections. However, since pathogens secrete a number of CWDEs at the onset of infection, we combined more inhibitors in a single wheat genotype to reinforce further the cell-wall barrier. We combined polygalacturonase (PG) inhibiting protein (PGIP) and pectin methyl esterase inhibitor (PMEI), both controlling the activity of PG, one of the first CWDEs secreted during infection. We also pyramided PGIP and TAXI-III, a xylanase inhibitor that controls the activity of xylanases, key factors for the degradation of xylan, a main component of cereal cell wall. We demonstrated that the pyramiding of PGIP and PMEI did not contribute to any further improvement of disease resistance. However, the presence of both pectinase inhibitors ensured a broader spectrum of disease resistance. Conversely, the PGIP and TAXI-III combination contributed to further improvement of Fusarium head blight (FHB) resistance, probably because these inhibitors target the activity of different types of CWDEs, i.e., PGs and xylanases. Worth mentioning, the reduction of FHB symptoms is accompanied by a reduction of deoxynivalenol accumulation with a foreseen great benefit to human and animal health.

Functional characterization and transient expression manipulation of a new sesquiterpene synthase involved in ß-caryophyllene accumulation in Ocimum.

The genus Ocimum has a unique blend of diverse secondary metabolites, with major proportion of terpenoids including mono- and sesquiterpenes. Although, ß-Caryophyllene, bicyclic sesquiterpene, is one of the major terpene found in Ocimum species and known to possess several biological activities, not much is known about its biosynthesis in Ocimum. Here, we describe isolation and characterization of ß-caryophyllene synthase gene from Ocimum kilimandscharicum Gürke (OkBCS- GenBank accession no. KP226502). The open reading frame of 1629 bp encoded a protein of 542 amino acids with molecular mass of 63.6 kDa and pI value of 5.66. The deduced amino acid sequence revealed 50-70% similarity with known sesquiterpene synthases from angiosperms. Recombinant OkBCS converted farnesyl diphosphate to ß-caryophyllene as a major product (94%) and 6% α-humulene. Expression variation of OkBCS well corroborated with ß-caryophyllene levels in different tissues from five Ocimum species. OkBCS transcript revealed higher expression in leaves and flowers. Further, agro-infiltration based transient expression manipulation with OkBCS over-expression and silencing confirmed its role in ß-caryophyllene biosynthesis. These findings may potentially be further utilized to improve plant defense against insect pests.

Insight into the role of grafting and arbuscular mycorrhiza on cadmium stress tolerance in tomato.

Physiological, biochemical, metabolite changes, and gene expression analysis of greenhouse tomato (Solanum lycopersicum L.) were investigated in two grafting combinations (self-grafted 'Ikram' and 'Ikram' grafted onto interspecific hybrid rootstock `Maxifort'), with and without arbuscular mycorrhizal (AM), exposed to 0 and 25 µM Cd. Tomato plants responded to moderate Cadmium (Cd) concentration by decreasing yield and crop growth parameters due to the accumulation of Cd in leaf tissue, inhibition of the PS II activity, reduced nutrients translocation, and also to the oxidative stress as evidenced by enhanced hydrogen peroxide (H2O2) generation, ion leakage, and lipid peroxidation. AM inoculation significantly enhanced the metal concentration in shoots and reduced growth and yield. The Ikram/Maxifort combination induced higher antioxidant enzymes, higher accumulation of proline and reduction of lipid peroxidation products. This suggests that the use of Maxifort rootstock in tomato has a high reactive oxygen species scavenging activity since lower H2O2 concentrations were observed in the presence of Cd. The higher crop performance of Ikram/Maxifort in comparison to Ikram/Ikram combination was also due to the improved nutritional status (higher P, K, Ca, Fe, Mn, and Zn) and increased availability of metabolites involved in cadmium tolerance (phytochelatin PC2, fructans, and inulins). The up-regulation of LeNRAMP3 gene in leaf of Ikram/Maxifort could explain the better nutritional status of interspecific grafting combination (higher Fe, Mn, and Zn).

An update on polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein that protects crop plants against pathogens.

Polygalacturonase inhibiting proteins (PGIPs) are cell wall proteins that inhibit the pectin-depolymerizing activity of polygalacturonases secreted by microbial pathogens and insects. These ubiquitous inhibitors have a leucine-rich repeat structure that is strongly conserved in monocot and dicot plants. Previous reviews have summarized the importance of PGIP in plant defense and the structural basis of PG-PGIP interaction; here we update the current knowledge about PGIPs with the recent findings on the composition and evolution of pgip gene families, with a special emphasis on legume and cereal crops. We also update the information about the inhibition properties of single pgip gene products against microbial PGs and the results, including field tests, showing the capacity of PGIP to protect crop plants against fungal, oomycetes and bacterial pathogens.

The pgip family in soybean and three other legume species: evidence for a birth-and-death model of evolution.

BACKGROUND: Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) plant cell wall glycoproteins involved in plant immunity. They are typically encoded by gene families with a small number of gene copies whose evolutionary origin has been poorly investigated. Here we report the complete characterization of the full complement of the pgip family in soybean (Glycine max [L.] Merr.) and the characterization of the genomic region surrounding the pgip family in four legume species. RESULTS: BAC clone and genome sequence analyses showed that the soybean genome contains two pgip loci. Each locus is composed of three clustered genes that are induced following infection with the fungal pathogen Sclerotinia sclerotiorum (Lib.) de Bary, and remnant sequences of pgip genes. The analyzed homeologous soybean genomic regions (about 126 Kb) that include the pgip loci are strongly conserved and this conservation extends also to the genomes of the legume species Phaseolus vulgaris L., Medicago truncatula Gaertn. and Cicer arietinum L., each containing a single pgip locus. Maximum likelihood-based gene trees suggest that the genes within the pgip clusters have independently undergone tandem duplication in each species. CONCLUSIONS: The paleopolyploid soybean genome contains two pgip loci comprised in large and highly conserved duplicated regions, which are also conserved in bean, M. truncatula and C. arietinum. The genomic features of these legume pgip families suggest that the forces driving the evolution of pgip genes follow the birth-and-death model, similar to that proposed for the evolution of resistance (R) genes of NBS-LRR-type.