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

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

ISOLATION AND CHARACTERIZATION OF XYLANASE INHIBITORS FROM CHROMOSOME GROUP 5 OF WHEAT

Cereals contain xylanase inhibitor proteins (XIs) which inhibit microbial xylanases from glycoside hydrolase families 10 and 11. In wheat, three types of XIs have been identified: Triticum aestivum XI (TAXI), xylanase inhibitor protein (XIP) and thaumatin-like XI (TLXI), and each of them is represented by a multiple number of genes. Several observations suggest that these inhibitors are important plant defence components. For example, XIs are effective against xylanases of microbial origin and not against plant xylanases. XIs share a significant sequence similarity with pathogenesis-related proteins (PR), are localized in the apoplastic region and are induced by wounding, jasmonic acid and pathogen infection. Moreover, the importance of xylaneses during pathogenesis has been demonstrated for the fungal pathogens Botrytis cinerea and Septoria tritici. Here we report the isolation and localization on chromosome group 5 of some XI genes. Moreover, to verify in planta the role of these XIs in wheat defence, we produced a number of transgenic plants over-expressing XipI, XipIII and TaxiIII. We are also attempting to silence TaxiIII and XipIII and a number of regenerated wheat plants have been obtained. The over-expression of XIs under control of the maize Ubi-1 promoter endows wheat with the capacity to produce these inhibitor constitutively and through in vitro inhibition assays using total protein extract from transgenic tissue we demonstrated the capacity of the transgenic XI to retain the inhibition properties against fungal xylanases. The transgenic wheat plants are under investigation to verify their response to the fungal pathogens Bipolaris sorokiniana and Fusarium graminearum

TRANSCRIPT ANALYSIS OF THE BEAN POLYGALACTURONASE INHIBITING PROTEIN GENE FAMILY REVEALS THAT PVPGIP2 IS EXPRESSED IN THE WHOLE PLANT AND IS STRONGLY INDUCED BY PATHOGEN INFECTION

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699International audienceThe expression analysis of the four polygalacturonase-inhibiting protein (Pgip) genes composing the bean (Phaseolus vulgaris L.) Pgip family showed a pattern of transcriptional variation in young leaves, hypocotyls, roots and pods with Pvpgip1 not expressed, Pvpgip2 expressed in all organs, Pvpgip3 and Pvpgip4 poorly expressed in roots. We compared also transcript accumulation of the four Pvpgip genes during infection of bean plants with the fungal pathogens Botrytis cinerea, Sclerotinia sclerotiorum and Colletotrichum demuthianum. qRT-PCR analyses showed that the transcript level of Pvpgip1, Pvpgip2 and Pvpgip3 increases significantly following fungal infection, whereas Pvpgip4 remains unchanged. The level of induction was different between the three genes, Pvpgip2 exhibiting the strongest transcript accumulation. The induction pattern was similar in the pathosystems bean-S. sclerotiorum, bean-B. cinerea, and in the compatible interaction bean-C. lindemuthianum, with a maximum of transcript accumulation in the late stage of infection. Instead, in the incompatible interaction bean-C. lindemuthianum, Pvpgip1, Pvpgip2 and Pvpgip3 showed an early and transient transcript accumulation, with Pvpgip2 exhibiting an earlier and higher induction. These results extend previous analyses of the whole Pup gip transcript and provide additional evidences of the relevant role of PvPGIP2 in plant defence

EXPRESSED PECTIN METHYLESTERASE INHIBITOR (PMEI) GENES SHOW A DIFFERENT PATTERN OF ACCUMULATION IN WHEAT TISSUE AND FOLLOWING FUNGAL INFECTION.

Cell wall pectin degradation represents an important step for a successful infection of the host tissue. An important feature of pectin is its degree and pattern of methyl esterification. Highly methyl esterified pectin or a random distribution of methyl ester can be associated with an increased host resistance response. Pectin methyl esterification is controlled by the activity of pectin methylesterase (PME), which de-methylate esterified pectin. Since the activity of PME can be controlled by its inhibitor protein (PMEI), we are characterizing Pmei genes in wheat to manipulate the methyl esterification of pectin and shed light on the involvement of this feature in wheat resistance. We report the characterization of the first Pmei gene (Tdpmei3) in wheat and the isolation of two additional Pmei-like genes (Tdpmei1 and Tdpmei2). qRT-PCR analysis showed that these genes are regulated during leaf development and Tdpmei1 and Tdpmei2 accumulate strongly in the ovary and stamen, whereas Tdpmei3 accumulate mainly in the stem. Tdpmei1 and Tdpmei3 are not induced following wheat leaf infection with the fungal pathogen Bipolaris sorokiniana, whereas Tdpmei2 transcript accumulate slightly

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, \u3b1-amylase/trypsin inhibitors (ATI) are involved in baker\u2019s 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

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

Recent Advances in Plant Nanobionics and Nanobiosensors for Toxicology Applications

Emerging applications in the field of nanotechnology are able to solve a gamut of problems surrounding the applications of agroecosystems and food technology. Nano Engineered Material (NEM) based nanosensors are important tools for monitoring plant signaling pathways and metabolism that are nondestructive, minimally invasive, and can provide real-time analysis of biotic and abiotic threats for better plant health. These sensors can measure chemical flux even at the single-molecule level. Therefore, plant health could be monitored through nutrient management, disease assessment, plant hormones level, environmental pollution, etc. This review provides a comprehensive account of the current trends and practices for the proposed NEM related research and its (i) structural aspect, (ii) experimental design and performance as well as (iii) mechanisms of field application in agriculture and food system. This review also discusses the possibility of integration of data from NEM based nanosensors in current and emerging trends of precision agriculture, urban farming, and plant nanobionics to adopt a sustainable approach in agriculture

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

Exploring the potential of engineering polygalacturonase‐inhibiting protein as an ecological, friendly, and nontoxic pest control agent

In plants, polygalacturonase-inhibiting proteins (PGIPs) play critical roles for resistance to fungal disease by inhibiting the pectin-depolymerizing activity of endopolygalacturonases (PGs), one type of enzyme secreted by pathogens that compromises plant cell walls and leaves the plant susceptible to disease. Here, the interactions between PGIPs from Phaseolus vulgaris (PvPGIP1 and PvPGIP2) and PGs from Aspergillus niger (AnPG2), Botrytis cinerea (BcPG1 and BcPG2), and Fusarium moniliforme (FmPG3) were reconstituted through a yeast two hybrid (Y2H) system to investigate the inhibition efficiency of various PvPGIP1 and 2 truncations and mutants. We found that tPvPGIP2_5-8, which contains LRR5 to LRR8 and is only one-third the size of the full length peptide, exhibits the same level of interactions with AnPG and BcPGs as the full length PvPGIP2 via Y2H. The inhibitory activities of tPvPGIP2_5-8 on the growth of A. niger and B. cinerea were then examined and confirmed on pectin agar. On pectin assays, application of both full length PvPGIP2 and tPvPGIP2_5-8 clearly slows down the growth of A. niger and B. cinerea. Investigation on the sequence-function relationships of PGIP utilizing a combination of site directed mutagenesis and a variety of peptide truncations suggests that LRR5 could have the most essential structural feature for the inhibitory activities, and may be a possible target for the future engineering of PGIP with enhanced activity. This study highlights the potential of plant-derived PGIPs as a candidate for future in planta evaluation as a pest control agent