North West Agriculture and Forestry University

Heat stress is a major abiotic stress that limits alfalfa (Medicago sativa L.) productivity. To test whether cytokinin-overexpressing engineered rhizobia could enhance heat tolerance, we inoculated alfalfa with modified rhizobia and evaluated their physiological and molecular responses. We found that inoculation with engineered rhizobia significantly increased trans-Zeatin content in alfalfa. Compared with control rhizobia-inoculated plants, engineered rhizobia-inoculated plants exhibited increased plant height, fresh/dry weight, relative water content, and photosynthetic efficiency (total chlorophyll and carotenoids), alongside reduced hydrogen peroxide (H₂O₂) and superoxide (O₂.-) levels under heat stress. RNA-seq analysis revealed that engineered rhizobia upregulated heat stress-responsive genes in alfalfa, which was further verified by qRT-PCR. Metabolomics analyses showed significant alterations in phenylpropanoid, flavonoid, phenolic acid, and salicylic acid metabolic pathways in engineered rhizobia-inoculated plants under heat stress. Contrary to conventional approaches, our results demonstrate that cytokinin-overexpressing rhizobia not only enhance alfalfa heat tolerance but also activate multi-pathway stress responses. Collectively, these findings propose a novel strategy for developing heat-tolerant alfalfa through engineered rhizobia-mediated cytokinin biosynthesis, which helps to promote the development of sustainable alfalfa breeding for heat-tolerant varieties.

Of the seven serotypes of foot-and-mouth disease virus (FMDV) strains circulating globally, serotype Asia1 has been effectively eradicated in China through systematic vaccination in livestock. The structural characteristics of serotype Asia1 may enhance its immunogenicity compared to other serotypes. Herein, we present a preliminary exploration of Asia1-binding B-cell receptor repertoire, containing 3571 clones, and identified 17 porcine-derived neutralizing monoclonal antibodies (pnAbs) from the top 33 high-frequency clonotypes. The majority of pnAbs (14/17) recognized the epitopes on VP2, with a common determinant at residue 72 (D) on the B-C loop; two pnAbs (2/17) recognized a novel epitope spanning VP2 and VP3; and the remaining one (1/17) bound to the C-terminus of VP1. Furthermore, the antigenic structures on VP2 and spanning VP2 and VP3 were respectively elucidated by determining the cryo-EM structures of FMDV serotype Asia1 in complexes with two pnAbs, PAS5 and PAS12. The light chain of PAS5, forming the majority of contact sites with the viral particle, focuses on the βB, B-C loop, βC and H-I loop of VP2, with key determinants at residues 68, 72 and 77 around the three-fold axis, corresponding to antigenic site 2. The contact sites of both VH and VL of PAS12 uncover a novel antigenic structure comprising the B-C, and H-I loops on VP2, and the B-B knob and βB on VP3, with key determinants at residue 73 on VP2 and 59 on VP3. Subsequently, site-directed competitive ELISA analysis of sera from primary and booster vaccinated pigs revealed a balanced antibody response profile, suggesting a potentially even immunodominance among antigenic site 2, VP1 G-H loop, and the novel antigenic structure spanning VP2 and VP3 on FMDV serotype Asia1. Compared to the focused immunodominance observed in other serotypes, this balanced antigenic recognition across VP1, VP2, and VP3 of FMDV serotype Asia1 reflects a diversified antibody response that may contribute to effective neutralization and protection.

Salinity threatens crop cultivation and productivity worldwide; however, to improve salinity tolerance in crop plants, foliar application of bio‐stimulants is thought to be a worthwhile technology. The current study was carried out to reveal the impact of foliar‐applied amino acids, phenylalanine (75 and 100 mg L −1 ) and methionine (25 and 50 mg L −1 ) on osmolyte accumulation, antioxidant activity, and yield of four cucumber genotypes (HC‐999, Valley, NSC‐CM1, and Akbar) under salt stress (100 mM NaCl). Regarding the individual effects, foliar spray of 50 mg L −1 methionine significantly enhanced the yield production of all cucumber genotypes; whereas foliar spray of phenylalanine at 100 mg L −1 significantly enhanced the antioxidant activity and osmolytes accumulation under salinity stress in comparison to other treatments. Combined amino acids (methionine at 25 mg L −1 and phenylalanine at 100 mg L −1 ) application significantly increased the fruit weight and antioxidant activity in saline conditions as compared to all other treatments. Among the genotypes, Valley was the most tolerant to salt stress as compared to the others. Overall, 25 mg L −1 methionine with 100 mg L −1 phenylalanine can be recommended for the farming community to avoid the yield loss of cucumber in salt‐affected soils.