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

Production and characterization of wheat lines silenced in alpha amylase/trypsin inhibitor genes involved in adverse reactions to wheat

Although wheat is the most consumed crop worldwide, it is also the main factor triggering different adverse reactions, among which celiac disease, true allergies and Non Celiac Wheat Sensitivity (NCWS). Among allergies, the so called \u201cbaker\u2019s asthma\u201d, is the most common professional asthma in Europe and is caused mainly by proteins present in the soluble fraction, especially alpha-amylase/trypsin inhibitors (ATI). Recent findings indicate in this class of proteins also the main factor triggering NCWS, that at present affects people with a frequency around 1:80, higher than celiac disease (1:100), but this is still a matter of debate. On this basis, we have produced RNAi wheat plants (both durum and bread wheat) in which different ATI genes have been silenced, to be used as a proof of concept, in order to test if they have a minor impact on adverse reactions, by using in vitro tests. We have silenced CM3, CM16 and 0.28 genes and have now available several lines in T4 generation. ELISA tests and immunoblotting analysis, by using a monoclonal antibody against ATI proteins, have shown that RNAi silenced wheat kernels present a lower amount of ATI proteins. Moreover, we are characterizing these lines in relation to respiratory allergies. Protein extracts from silenced plants are being tested by using human sera of allergic patients in order to verify if a lower amount of immunogenic polypeptides is recognized in comparison to wild type untransformed plants. If this is the case, the realization of new wheat genotypes expressing a lower amount of ATI proteins can be a realistic target to be reached by classical breeding procedures

Silencing of ATI genes involved in adverse reactions to wheat by RNAi and CRISPR-Cas9 technologies

Although wheat is consumed worldwide as a staple food, it can give rise to different adverse reactions, some of which have not been deeply characterized. They are caused mainly by wheat proteins, both gluten and non-gluten proteins. Structural and metabolic proteins, like \u3b1amylase/trypsin inhibitors (ATI) are involved in the onset of wheat allergies (bakers\u2019 asthma) and probably non-coeliac wheat sensitivity (NCWS). The ATI are encoded by a multigene family dispersed over several. Notably, WTAI-CM3 and WTAI-CM16 subunits are involved in the onset of bakers\u2019 asthma and are likely to contribute to NCWS. In this study we report the RNAi silencing of WTAI-CM3, WTAI-CM16 and WMAI-0.28 genes in the bread wheat cultivar Bobwhite and the CRISPR/Cas9 mediated gene knockout of WTAI-CM3 and WTAI-CM16 in the durum wheat cultivar Svevo. We have obtained different RNAi transgenic lines showing an effective decrease in the expression in the targeted genes. These lines do not show differences in terms of yield, but have unintended effects on the accumulation of the high molecular weight glutenin subunits which play a crucial role in the technological performances of wheat flour. Furthermore, the editing of WTAI-CM3 and WTAI-CM16 genes was obtained through a CRISPR-Cas9 multiplexing strategy in the Italian durum wheat cultivar Svevo with a marker-free approach. The regeneration of plants without selection agents allowed T0 homozygous mutant plants to be obtained without the integration in the wheat genome of CRISPR/Cas9 vectors, demonstrating the capability of CRISPR technology to produce wheat lines in a reduced time compared to conventional breeding approaches. The possibility to develop new wheat genotypes accumulating a lower amount of proteins effectively involved in such pathologies, not only offers the possibility to use them as a basis for the creation of wheat varieties with a lower impact on adverse reactions, but also to test if these proteins are actually implicated in those pathologies for which the triggering factor has yet to be established

Wheat ati cm3, cm16 and 0.28 allergens produced in pichia pastoris display a different eliciting potential in food allergy to wheat

International audienceAlthough 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 beta-hexosaminidase release observed for CM3 protein

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

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

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