Prospects for developing allergen-depleted food crops.

In addition to the challenge of meeting global demand for food production, there are increasing concerns about food safety and the need to protect consumer health from the negative effects of foodborne allergies. Certain bio-molecules (usually proteins) present in food can act as allergens that trigger unusual immunological reactions, with potentially life-threatening consequences. The relentless working lifestyles of the modern era often incorporate poor eating habits that include readymade prepackaged and processed foods, which contain additives such as peanuts, tree nuts, wheat, and soy-based products, rather than traditional home cooking. Of the predominant allergenic foods (soybean, wheat, fish, peanut, shellfish, tree nuts, eggs, and milk), peanuts (Arachis hypogaea) are the best characterized source of allergens, followed by tree nuts (Juglans regia, Prunus amygdalus, Corylus avellana, Carya illinoinensis, Anacardium occidentale, Pistacia vera, Bertholletia excels), wheat (Triticum aestivum), soybeans (Glycine max), and kidney beans (Phaseolus vulgaris). The prevalence of food allergies has risen significantly in recent years including chance of accidental exposure to such foods. In contrast, the standards of detection, diagnosis, and cure have not kept pace and unfortunately are often suboptimal. In this review, we mainly focus on the prevalence of allergies associated with peanut, tree nuts, wheat, soybean, and kidney bean, highlighting their physiological properties and functions as well as considering research directions for tailoring allergen gene expression. In particular, we discuss how recent advances in molecular breeding, genetic engineering, and genome editing can be used to develop potential low allergen food crops that protect consumer health.

A Perspective Review on Understanding Drought Stress Tolerance in Wild Banana Genetic Resources of Northeast India.

The enormous perennial monocotyledonous herb banana (Musa spp.), which includes dessert and cooking varieties, is found in more than 120 countries and is a member of the order Zingiberales and family Musaceae. The production of bananas requires a certain amount of precipitation throughout the year, and its scarcity reduces productivity in rain-fed banana-growing areas due to drought stress. To increase the tolerance of banana crops to drought stress, it is necessary to explore crop wild relatives (CWRs) of banana. Although molecular genetic pathways involved in drought stress tolerance of cultivated banana have been uncovered and understood with the introduction of high-throughput DNA sequencing technology, next-generation sequencing (NGS) techniques, and numerous "omics" tools, unfortunately, such approaches have not been thoroughly implemented to utilize the huge potential of wild genetic resources of banana. In India, the northeastern region has been reported to have the highest diversity and distribution of Musaceae, with more than 30 taxa, 19 of which are unique to the area, accounting for around 81% of all wild species. As a result, the area is regarded as one of the main locations of origin for the Musaceae family. The understanding of the response of the banana genotypes of northeastern India belonging to different genome groups to water deficit stress at the molecular level will be useful for developing and improving drought tolerance in commercial banana cultivars not only in India but also worldwide. Hence, in the present review, we discuss the studies conducted to observe the effect of drought stress on different banana species. Moreover, the article highlights the tools and techniques that have been used or that can be used for exploring and understanding the molecular basis of differentially regulated genes and their networks in different drought stress-tolerant banana genotypes of northeast India, especially wild types, for unraveling their potential novel traits and genes.

Midgut aminopeptidase N expression profile in castor semilooper (Achaea janata) during sublethal Cry toxin exposure.

The midgut of lepidopteran larvae is a multifunctional tissue that performs roles in digestion, absorption, immunity, transmission of pathogens and interaction with ingested various molecules. The proteins localized at the inner apical brush border membrane are primarily digestive proteases, but some of them, like aminopeptidase N, alkaline phosphatase, cadherins, ABC transporter C2, etc., interact with Crystal (Cry) toxins produced by Bacillus thuringiensis (Bt). In the present study, aminopeptidase N (APN) was characterized as Cry-toxin-interacting protein in the larval midgut of castor semilooper, Achaea janata. Transcriptomic and proteomic analyses revealed the presence of multiple isoforms of APNs (APN1, 2, 4, 6 and 9) which have less than 40% sequence similarity but show the presence of characteristic 'GAMENEG' and zinc-binding motifs. Feeding a sublethal dose of Cry toxin caused differential expression of various APN isoform. Further, 6thgeneration Cry-toxin-exposed larvae showed reduced expression of APN2. This report suggests that A. janata larvae exploit altered expression of APNs to overcome the deleterious effects of Cry toxicity, which might facilitate toxin tolerance in the long run.

Response of Midgut Trypsin- and Chymotrypsin-Like Proteases of Helicoverpa armigera Larvae Upon Feeding With Peanut BBI: Biochemical and Biophysical Characterization of PnBBI.

Proteinase/Protease inhibitors (PIs) from higher plants play an important role in defense and confer resistance against various insect pests and pathogens. In the present study, Bowman-Birk Inhibitor (BBI) was purified from mature seeds of an interspecific advanced hybrid peanut variety (4368-1) using chromatographic techniques. The biochemical and biophysical characteristics such as low molecular mass, presence of several isoinhibitors and higher-ordered dimer/tetramer, predominance of antiparallel ß-sheets and random coils in secondary structure, reactive sites against trypsin and chymotrypsin, broad spectrum of stability toward extreme pH and temperature along with MALDI TOF-TOF analysis (ProteomeXchange identifier PXD016933) ascertained the purified biomolecule from peanut as BBI (PnBBI). Surface plasmon resonance competitive binding analysis revealed the bifunctional PnBBI is a trypsin specific inhibitor with 1:2 stoichiometry as compared to chymotrypsin. A concentration-dependent self-association tendency of PnBBI was further confirmed by 'red shift' in the far-UV CD spectra. Furthermore, the insecticidal potential of PnBBI against Helicoverpa armigera was assessed by in vitro assays and in vivo feeding experiments. A significant reduction in larval body weight was observed with concomitant attenuation in the activity of midgut trypsin-like proteases of H. armigera (HaTPs) fed on PnBBI supplemented diet. The one and two-dimensional zymography studies revealed the disappearance of several isoforms of HaTP upon feeding with PnBBI. qRT-PCR analysis further suggests the role of PnBBI in not only inhibiting the activity of midgut trypsin and chymotrypsin-like proteases but also in modulating their expression. Taken together, the results provide a biochemical and molecular basis for introgressed resistance in peanut interspecific advanced hybrid variety against H. armigera.

Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea.

Rhynchosia sublobata, a wild relative of pigeonpea, possesses defensive proteinase/protease inhibitors (PIs). Characterization of trypsin specific PIs (RsPI) separated from seeds by column chromatography using 2-D gel electrophoresis and Edman degradation method identified R. sublobata possessed both Bowman-Birk isoinhibitors (RsBBI) and Kunitz isoinhibitors (RsKI). A quick method was developed to separate RsBBI and RsKI from RsPI based on their differential solubility in TCA and acetate buffer. N-terminus sequencing of RsBBI and RsKI by MALDI-ISD ascertained the presence of Bowman Birk and Kunitz type isoinhibitors in R. sublobata. RsBBI (9216 Da) and RsKI (19,412 Da) exhibited self-association pattern as revealed by western blotting with anti-BBI antibody and MALDI-TOF peptide mass fingerprint analysis, respectively. RsBBI and RsKI varied significantly in their biochemical, biophysical and insecticidal properties. RsBBI inhibited the activity of trypsin (Ki = 128.5 ±â€¯4.5 nM) and chymotrypsin (Ki = 807.8 ±â€¯23.7 nM) while RsKI (Ki = 172.0 ±â€¯9.2 nM) inhibited the activity of trypsin alone, by non-competitive mode. The trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) activities of RsBBI were stable up to 100 °C. But, RsBBI completely lost its TI and CI activities on reduction with 3 mM DTT. Conversely, RsKI lost its TI activity on heating at 100 °C and retained >60% of its TI activity in presence of 3 mM DTT. CD spectroscopic studies on RsBBI and RsKI showed their secondary structural elements in the following order: random coils > ß-sheets/ß-turns > α-helix. However, RsKI showed reversible denaturation midpoint (Tm) of 75 °C. Further, the significant inhibitory activity of RsBBI (IC50 = 24 ng) and RsKI (IC50 = 59 ng) against trypsin-like gut proteases of Achaea janata (AjGPs) and Helicoverpa armigera (HaGPs) suggest them as potential biomolecules in the management of A. janata and H. armigera, respectively.

Purification and Partial Characterization of Trypsin-Specific Proteinase Inhibitors from Pigeonpea Wild Relative Cajanus platycarpus L. (Fabaceae) Active against Gut Proteases of Lepidopteran Pest Helicoverpa armigera.

Proteinase inhibitors (PIs) are natural defense proteins of plants found to be active against gut proteases of various insects. A pigeonpea wild relative Cajanus platycarpus was identified as a source of resistance against Helicoverpa armigera, a most devastating pest of several crops including pigeonpea. In the light of earlier studies, trypsin-specific PIs (CpPI 63) were purified from mature dry seeds of C. platycarpus (ICPW-63) and characterized their biochemical properties in contributing to H. armigera resistance. CpPI 63 possessed significant H. armigera gut trypsin-like proteinase inhibitor (HGPI) activity than trypsin inhibitor (TI) activity. Analysis of CpPI 63 using two-dimensional (2-D) electrophoresis and matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that it contained several isoinhibitors and small oligomers with masses ranging between 6 and 58 kDa. The gelatin activity staining studies suggest that these isoinhibitors and oligomers possessed strong inhibitory activity against H. armigera gut trypsin-like proteases (HGPs). The N-terminal sequence of the isoinhibitors (pI 6.6 and pI 5.6) of CpPI 63 exhibited 80% homology with several Kunitz trypsin inhibitors (KTIs) as well as miraculin-like proteins (MLPs). Further, modification of lysine residue(s) lead to 80% loss in both TI and HGPI activities of CpPI 63. In contrast, the TI and HGPI activities of CpPI 63 were stable over a wide range of temperature and pH conditions. The reported results provide a biochemical basis for pod borer resistance in C. platycarpus.

Structural and functional characterization of proteinase inhibitors from seeds of Cajanus cajan (cv. ICP 7118).

Proteinase inhibitors (C11PI) from mature dry seeds of Cajanus cajan (cv. ICP 7118) were purified by chromatography which resulted in 87-fold purification and 7.9% yield. SDS-PAGE, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF) mass spectrum and two-dimensional (2-D) gel electrophoresis together resolved that C11PI possessed molecular mass of 8385.682 Da and existed as isoinhibitors. However, several of these isoinhibitors exhibited self association tendency to form small oligomers. All the isoinhibitors resolved in Native-PAGE and 2-D gel electrophoresis showed inhibitory activity against bovine pancreatic trypsin and chymotrypsin as well as Achaea janata midgut trypsin-like proteases (AjPs), a devastating pest of castor plant. Partial sequences of isoinhibitor (pI 6.0) obtained from MALDI-TOF/TOF analysis and N-terminal sequencing showed 100% homology to Bowman-Birk Inhibitors (BBIs) of leguminous plants. C11PI showed non-competitive inhibition against trypsin and chymotrypsin. A marginal loss (<15%) in C11PI activity against trypsin at 80 (°)C and basic pH (12.0) was associated with concurrent changes in its far-UV CD spectra. Further, in vitro assays demonstrated that C11PI possessed significant inhibitory potential (IC50 of 78 ng) against AjPs. On the other hand, in vivo leaf coating assays demonstrated that C11PI caused significant mortality rate with concomitant reduction in body weight of both larvae and pupae, prolonged the duration of transition from larva to pupa along with formation of abnormal larval-pupal and pupal-adult intermediates. Being smaller peptides, it is possible to express C11PI in castor to protect them against its devastating pest A. janata.