Background Allium sativum leaf agglutinin (ASAL) is a 25-kDa homodimeric, insecticidal, mannose binding lectin whose subunits are assembled by the C-terminal exchange process. An attempt was made to convert dimeric ASAL into a monomeric form to correlate the relevance of quaternary association of subunits and their functional specificity. Using SWISS-MODEL program a stable monomer was designed by altering five amino acid residues near the C-terminus of ASAL.Methodology/Principal FindingsBy introduction of 5 site-specific mutations (-DNSNN-), a β turn was incorporated between the 11th and 12th β strands of subunits of ASAL, resulting in a stable monomeric mutant ASAL (mASAL). mASAL was cloned and subsequently purified from a pMAL-c2X system. CD spectroscopic analysis confirmed the conservation of secondary structure in mASAL. Mannose binding assay confirmed that molecular mannose binds efficiently to both mASAL and ASAL. In contrast to ASAL, the hemagglutination activity of purified mASAL against rabbit erythrocytes was lost. An artificial diet bioassay of Lipaphis erysimi with mASAL displayed an insignificant level of insecticidal activity compared to ASAL. Fascinatingly, mASAL exhibited strong antifungal activity against the pathogenic fungi Fusarium oxysporum, Rhizoctonia solani and Alternaria brassicicola in a disc diffusion assay. A propidium iodide uptake assay suggested that the inhibitory activity of mASAL might be associated with the alteration of the membrane permeability of the fungus. Furthermore, a ligand blot assay of the membrane subproteome of R. solani with mASAL detected a glycoprotein receptor having interaction with mASAL.Conclusions/SignificanceConversion of ASAL into a stable monomer resulted in antifungal activity. From an evolutionary aspect, these data implied that variable quaternary organization of lectins might be the outcome of defense-related adaptations to diverse situations in plants. Incorporation of mASAL into agronomically-important crops could be an alternative method to protect them from dramatic yield losses from pathogenic fungi in an effective manner.

Rice, the major food crop of world is severely affected by homopteran sucking pests. We introduced coding sequence of Allium sativum leaf agglutinin, ASAL, in rice cultivar IR64 to develop sustainable resistance against sap-sucking planthoppers as well as eliminated the selectable antibiotic-resistant marker gene hygromycin phosphotransferase (hpt) exploiting cre/lox site-specific recombination system. An expression vector was constructed containing the coding sequence of ASAL, a potent controlling agent against green leafhoppers (GLH, Nephotettix virescens) and brown planthopper (BPH, Nilaparvata lugens). The selectable marker (hpt) gene cassette was cloned within two lox sites of the same vector. Alongside, another vector was developed with chimeric cre recombinase gene cassette. Reciprocal crosses were performed between three single-copy T(0) plants with ASAL- lox-hpt-lox T-DNA and three single-copy T(0) plants with cre-bar T-DNA. Marker gene excisions were detected in T(1) hybrids through hygromycin sensitivity assay. Molecular analysis of T(1) plants exhibited 27.4% recombination efficiency. T(2) progenies of L03C04(1) hybrid parent showed 25% cre negative ASAL-expressing plants. Northern blot, western blot and ELISA showed significant level of ASAL expression in five marker-free T(2) progeny plants. In planta bioassay of GLH and BPH performed on these T(2) progenies exhibited radical reduction in survivability and fecundity compared with the untransformed control plants.

This study reports the root endophytic microbial community profile in rice (Oryza sativa L.), the largest food crop of Asia, using 16S rRNA gene amplicon sequencing. Metagenome of OS01 and OS04 consisted of 11,17,900 sequences with 300 Mbp size and average 55.6% G + C content. Data of this study are available at NCBI Bioproject (PRJNA360379). The taxonomic analysis of 843 OTU's showed that the sequences belonged to four major phyla revealing dominance of Proteobacteria, Firmicutes, Cyanobacteria and Actinobacteria. Results reveal the dominance of Bacillus as major endophytic genera in rice roots, probably playing a key role in Nitrogen fixation.

Root endophytes are considered to be one of the potent environment-friendly substitutes for chemical fertilizers, as they possess an ability to induce crosstalk inside the hosts for growth promotion, nitrogen fixation, phosphate solubilization and iron sequestration. This study aimed to explore and evaluate the key root endophytic bacterial consortia of two widely cultivated varieties of rice (Oryza sativa L.), cv. ‘Saraswati’ (OS01) and cv. ‘Kunti’ (OS04). Detailed comparative metagenome data were generated for endophytes of OS01 and OS04 and the species richness was calculated. OS01 showed higher endophyte species richness than OS04, with alpha diversity values of 3.10 and 2.40, respectively. Bacillus, Magnetospirillum, Methanocystis, Desulfomicrobium and Pantoea were identified as common endophyte members for both cultivars. Solibacillus, Paenibacillus, Candidatus, and Melospira were unique members of OS01, and Herbaspirillum, Pandoraea, Anabaenopsis for OS04. Considerable occurrence of nitrogen fixing bacteria and methanogenic bacteria in the cultivars confirmed biological nitrogen fixation, which can contribute to plant development. Core homeotic pathways of amino acid biosynthesis and carbon metabolism were also reflected in endophytes from both cultivars, indicating a supportive environment for microorganisms. Sulfur metabolism pathways were likewise predicted to be active in the niche under study, which may be attributed as a response to arsenic stress. Furthermore, the most abundant genera identified may potentially serve as crucial consortium candidates for host plant development and contribute to better yield in a sustainable manner.