Genome-Wide Investigation of the Cysteine Synthase Gene Family Shows That Overexpression of CSase Confers Alkali Tolerance to Alfalfa (Medicago sativa L.)

Alfalfa is widely grown worldwide as a perennial high-quality legume forage and as a good ecological landcover. The cysteine synthase (CSase) gene family is actively involved in plant growth and development and abiotic stress resistance but has not been systematically investigated in alfalfa. We identified 39 MsCSase genes on 4 chromosomes of the alfalfa genome. Phylogenetic analysis demonstrated that these genes were clustered into six subfamilies, and members of the same subfamily had similar physicochemical properties and sequence structures. Overexpression of the CSase gene in alfalfa increased alkali tolerance. Compared with control plants, the overexpression lines presented higher proline, soluble sugars, and cysteine and reduced glutathione contents and superoxide dismutase and peroxidase activities as well as lower hydrogen peroxide and superoxide anion contents after alkali stress. The relative expression of γ-glutamyl cysteine synthetase gene (a downstream gene of CSase) in the overexpression lines was much higher than that in the control line. The CSase gene enhanced alkalinity tolerance by regulating osmoregulatory substances and improving antioxidant capacity. These results provide a reference for studying the CSase gene family in alfalfa and expanding the alkali tolerance gene resources of forage plants.

Comparative Analysis of Coding and Non-Coding Features within Insect Tolerance Loci in Wheat with Their Homologs in Cereal Genomes

Food insecurity and malnutrition have reached critical levels with increased human population, climate fluctuations, water shortage; therefore, higher-yielding crops are in the spotlight of numerous studies. Abiotic factors affect the yield of staple food crops; among all, wheat stem sawfly (Cephus cinctus Norton) and orange wheat blossom midge (Sitodiplosis mosellana) are two of the most economically and agronomically harmful insect pests which cause yield loss in cereals, especially in wheat in North America. There is no effective strategy for suppressing this pest damage yet, and only the plants with intrinsic tolerance mechanisms such as solid stem phenotypes for WSS and antixenosis and/or antibiosis mechanisms for OWBM can limit damage. A major QTL and a causal gene for WSS resistance were previously identified in wheat, and 3 major QTLs and a causal gene for OWBM resistance. Here, we present a comparative analysis of coding and non-coding features of these loci of wheat across important cereal crops, barley, rye, oat, and rice. This research paves the way for our cloning and editing of additional WSS and OWBM tolerance gene(s), proteins, and metabolites.

Selection of the Salt Tolerance Gene GmSALT3 During Six Decades of Soybean Breeding in China

Salt tolerance is an important trait that affects the growth and yield of plants growing in saline environments. The salt tolerance gene GmSALT3 was cloned from the Chinese soybean cultivar Tiefeng 8, and its variation evaluated in Chinese wild soybeans and landraces. However, the potential role of GmSALT3 in cultivation, and its genetic variation throughout the history of Chinese soybean breeding, remains unknown. Here we identified five haplotypes of GmSALT3 in 279 Chinese soybean landraces using a whole genome resequencing dataset. Additionally, we developed five PCR-based functional markers: three indels and two cleaved amplified polymorphic sequences (CAPS) markers. A total of 706 Chinese soybean cultivars (released 1956–2012), and 536 modern Chinese breeding lines, were genotyped with these markers. The Chinese landraces exhibited relatively high frequencies of the haplotypes H1, H4, and H5. H1 was the predominant haplotype in both the northern region (NR) and Huanghuai region (HHR), and H5 and H4 were the major haplotypes present within the southern region (SR). In the 706 cultivars, H1, H2, and H5 were the common haplotypes, while H3 and H4 were poorly represented. Historically, H1 gradually decreased in frequency in the NR but increased in the HHR; while the salt-sensitive haplotype, H2, increased in frequency in the NR during six decades of soybean breeding. In the 536 modern breeding lines, H2 has become the most common haplotype in the NR, while H1 has remained the highest frequency haplotype in the HHR, and H5 and H1 were highest in the SR. Frequency changes resulting in geographically favored haplotypes indicates that strong selection has occurred over six decades of soybean breeding. Our molecular markers could precisely identify salt tolerant (98.9%) and sensitive (100%) accessions and could accurately trace the salt tolerance gene in soybean pedigrees. Our study, therefore, not only identified effective molecular markers for use in soybean, but also demonstrated how these markers can distinguish GmSALT3 alleles in targeted breeding strategies for specific ecoregions.

Identification and Analysis of Small Interfering RNAs Associated With Heat Stress in Flowering Chinese Cabbage Using High-Throughput Sequencing

Endogenous small interfering RNAs (siRNAs) are substantial gene regulators in eukaryotes and play key functions in plant development and stress tolerance. Among environmental factors, heat is serious abiotic stress that severely influences the productivity and quality of flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee). However, how siRNAs are involved in regulating gene expression during heat stress is not fully understood in flowering Chinese cabbage. Combining bioinformatical and next-generation sequencing approaches, we identified heat-responsive siRNAs in four small RNA libraries of flowering Chinese cabbage using leaves collected at 0, 1, 6, and 12 h after a 38°C heat-stress treatment; 536, 816, and 829 siRNAs exhibited substantial differential expression at 1, 6, and 12 h, respectively. Seventy-five upregulated and 69 downregulated differentially expressed siRNAs (DE-siRNAs) were common for the three time points of heat stress. We identified 795 target genes of DE-siRNAs, including serine/threonine-protein kinase SRK2I, CTR1-like, disease resistance protein RML1A-like, and RPP1, which may play a role in regulating heat tolerance. Gene ontology showed that predictive targets of DE-siRNAs may have key roles in the positive regulation of biological processes, organismal processes, responses to temperature stimulus, signaling, and growth and development. These novel results contribute to further understanding how siRNAs modulate the expression of their target genes to control heat tolerance in flowering Chinese cabbage.

Validation of Drought Tolerance Gene-linked Microsatellite Markers and Their Efficiency for Diversity Assessment in a Set of Soybean Genotypes

Aim: Soybean is well-thought-out to be a major crop owing to its significant involvement as vegetable oil and protein in human diet. However, inopportunely, its production has been melodramatically declined attributable to the commonness of drought related stress. Study Design: During the present study a total of 53 soybean genotypes were selected. For molecular diversity analysis as well as validation total 12 SSR markers were used. Molecular screening of soybean genotypes was done to determine the efficiency of available markers in genetic diversity analysis as well as their validation on the basis of their association with drought tolerance gene. Place and Duration of the Study: The present study was conducted at Department of Plant Molecular Biology and Biotechnology, College of Agriculture, Gwalior, Rajmata Vijayaraje Scindia Krishi Vishwa Vidyalaya, Gwalior, M.P., India during the year 2018 - 2019. Methodology: Template DNA of all 53 selected soybean genotypes extracted for molecular screening. The current investigation has been accomplished to validate the available SSR markers with their efficiency in genetic diversity analysis in a set of soybean genotypes. Results: Among applied drought tolerance gene-linked 12 SSR molecular markers, the highest genetic diversity (0.6629) was noticed in Satt520 while lowest (0.0370) was in Satt557 with an average of 0. 3746.While, the highest PIC value was 0.5887 prearranged by Satt520 and lowest 0.0363 by Satt557 with the mean worth of 0.3063. Conclusion: Dendrogram constructed on the basis of banding profile of employed markers was able to discriminate some putative drought tolerant genotypes i.e., JS97-52, JS95-60 from rest of the genotypes. The results of the present examination may donate towards enhancement of soybean genotypes to bread drought tolerant varieties.

Heat Stress Tolerance Gene FpHsp104 Affects Conidiation and Pathogenicity of Fusarium pseudograminearum

Heat shock protein Hsp104, a homolog of the bacterial chaperone ClpB and plant Hsp100, plays an essential part in the response to heat and various chemical agents in Saccharomyces cerevisiae. However, their functions remain largely unknown in plant fungal pathogens. Here, we report the identification and functional characterization of a plausible ortholog of yeast Hsp104 in Fusarium pseudograminearum, which we termed FpHsp104. Deletion mutant of FpHsp104 displayed severe defects in the resistance of heat shock during F. pseudograminearum mycelia and conidia when exposed to extreme heat. We also found that the protein showed dynamic localization to small particles under high temperature. However, no significant differences were detected in osmotic, oxidative, or cell wall stress responses between the wild-type and Δfphsp104 strains. Quantitative real-time PCR analysis showed that FpHsp104 was upregulated in the conidia, and disruption of FpHsp104 gene resulted in defects in conidia production, morphology, and germination. The transcript levels of conidiation-related genes of FpFluG, FpVosA, FpWetA, and FpAbaA were reduced in the Δfphsp104 mutant vs. the wild-type strain, but heat-shocked mRNA splicing repair was not affected in Δfphsp104. Moreover, Δfphsp104 mutant also showed attenuated virulence, but its DON synthesis was normal. These data from the first study of Hsp104 in F. pseudograminearum strongly suggest that FpHsp104 gene is an important element in the heat tolerance, development, and pathogenicity processes of F. pseudograminearum.

Cbl-b deficiency prevents functional but not phenotypic T cell anergy

T cell anergy is an important peripheral tolerance mechanism. We studied how T cell anergy is established using an anergy model in which the Zap70 hypermorphic mutant W131A is coexpressed with the OTII TCR transgene (W131AOTII). Anergy was established in the periphery, not in the thymus. Contrary to enriched tolerance gene signatures and impaired TCR signaling in mature peripheral CD4 T cells, CD4SP thymocytes exhibited normal TCR signaling in W131AOTII mice. Importantly, the maintenance of T cell anergy in W131AOTII mice required antigen presentation via MHC-II. We investigated the functional importance of the inhibitory receptor PD-1 and the E3 ubiquitin ligases Cbl-b and Grail in this model. Deletion of each did not affect expression of phenotypic markers of anergic T cells or T reg numbers. However, deletion of Cbl-b, but not Grail or PD-1, in W131AOTII mice restored T cell responsiveness and signaling. Thus, Cbl-b plays an essential role in the establishment and/or maintenance of unresponsiveness in T cell anergy.

Random Transfer of Ogataea polymorpha Genes into Saccharomyces cerevisiae Reveals a Complex Background of Heat Tolerance

Horizontal gene transfer, a process through which an organism acquires genes from other organisms, is a rare evolutionary event in yeasts. Artificial random gene transfer can emerge as a valuable tool in yeast bioengineering to investigate the background of complex phenotypes, such as heat tolerance. In this study, a cDNA library was constructed from the mRNA of a methylotrophic yeast, Ogataea polymorpha, and then introduced into Saccharomyces cerevisiae. Ogataea polymorpha was selected because it is one of the most heat-tolerant species among yeasts. Screening of S. cerevisiae populations expressing O. polymorpha genes at high temperatures identified 59 O. polymorpha genes that contribute to heat tolerance. Gene enrichment analysis indicated that certain S. cerevisiae functions, including protein synthesis, were highly temperature-sensitive. Additionally, the results confirmed that heat tolerance in yeast is a complex phenotype dependent on multiple quantitative loci. Random gene transfer would be a useful tool for future bioengineering studies on yeasts.