Molecular Mapping of a Gene for Fertility Restoration of Wild Abortive (WA) Cytoplasmic Male Sterility using a Basmati Rice Restorer Line

The inheritance and molecular mapping of a fertility restorer gene in basmati quality restorer line PRR-78 was carried out using an F₂ mapping population from the cross IR58025A X PRR-78 employing microsatellite markers. Dominant monogenic control of fertility restoration was observed in the F₂, and further confirmed by test cross data. Out of 44 sequence tagged microsatellite (STMS) markers used in the bulked segregant analysis (BSA), four differentiated the fertile bulk from the sterile bulk as well as the two parental lines from each other. One of these markers, RM258 located on chromosome 10, was found linked to the restorer gene at a distance of9.5 cM. Considering the RM258 location, additional STMS (RM171 and RM294A) and sequence tagged site (STS) primers derived from restriction fragment length polymorphic (RFLP) clones (G2155 and C1361) linked to fertility restorer gene(s) in other populations, were also used to find out a marker more tightly linked to the restorer gene. However, of these, RM171, RM294A and G2155 based primers amplified monomorphic fragments between parental lines and no amplification was observed with C1361. Cleaved amplified polymorphic sequence (CAPS) analysis of non-polymorphic STMS and STS markers and random amplified polymorphic DNA (RAPD) analysis using five random primers reportedly linked to restorer gene in other populations, also failed to differentiate the two parents. While, the marker RM258 is being used in the restorer breeding to identify putative restorer lines, search for additional tightly linked markers is underway.     

Monoclonal Antibodies against the Fusion Peptide of Hemagglutinin Protect Mice from Lethal Influenza A Virus H5N1 Infection

The HA2 glycopolypeptide (gp) is highly conserved in all influenza A virus strains, and it is known to play a major role in the fusion of the virus with the endosomal membrane in host cells during the course of viral infection. Vaccines and therapeutics targeting this HA2 gp could induce efficient broad-spectrum immunity against influenza A virus infections. So far, there have been no studies on the possible therapeutic effects of monoclonal antibodies (MAbs), specifically against the fusion peptide of hemagglutinin (HA), upon lethal infections with highly pathogenic avian influenza (HPAI) H5N1 virus. We have identified MAb 1C9, which binds to GLFGAIAGF, a part of the fusion peptide of the HA2 gp. We evaluated the efficacy of MAb 1C9 as a therapy for influenza A virus infections. This MAb, which inhibited cell fusion in vitro when administered passively, protected 100% of mice from challenge with five 50% mouse lethal doses of HPAI H5N1 influenza A viruses from two different clades. Furthermore, it caused earlier clearance of the virus from the lung. The influenza virus load was assessed in lung samples from mice challenged after pretreatment with MAb 1C9 (24 h prior to challenge) and from mice receiving early treatment (24 h after challenge). The study shows that MAb 1C9, which is specific to the antigenically conserved fusion peptide of HA2, can contribute to the cross-clade protection of mice infected with H5N1 virus and mediate more effective recovery from infection.Highly pathogenic avian influenza (HPAI) virus H5N1 strains are currently causing major morbidity and mortality in poultry populations across Asia, Europe, and Africa and have caused 385 confirmed human infections, with a fatality rate of 63.11% (37, 39). Preventive and therapeutic measures against circulating H5N1 strains have received a lot of interest and effort globally to prevent another pandemic outbreak. Influenza A virus poses a challenge because it rapidly alters its appearance to the immune system by antigenic drift (mutating) and antigenic shift (exchanging its components) (5). The current strategies to combat influenza include vaccination and antiviral drug treatment, with vaccination being the preferred option. The annual influenza vaccine aims to stimulate the generation of anti-hemagglutinin (anti-HA) neutralizing antibodies, which confer protection against homologous strains. Current vaccines have met with various degrees of success (31). The facts that these strategies target the highly variable HA determinant and that predicting the major HA types that pose the next epidemic threat is difficult are significant limitations to the current antiviral strategy. In the absence of an effective vaccine, therapy is the mainstay of control of influenza virus infection.Therefore, therapeutic measures against influenza will play a major role in case a pandemic arises due to H5N1 strains. Currently licensed antiviral drugs include the M2 ion-channel inhibitors (rimantidine and amantidine) and the neuraminidase inhibitors (oseltamivir and zanamivir). The H5N1 viruses are known to be resistant to the M2 ion-channel inhibitors (2, 3). Newer strains of H5N1 viruses are being isolated which are also resistant to the neuraminidase inhibitors (oseltamivir and zanamivir) (5, 17). The neuraminidase inhibitors also require high doses and prolonged treatment (5, 40), increasing the likelihood of unwanted side effects. Hence, alternative strategies for treatment of influenza are warranted.Recently, passive immunotherapy using monoclonal antibodies (MAbs) has been viewed as a viable option for treatment (26). The HA gene is the most variable gene of the influenza virus and also the most promising target for generating antibodies. It is synthesized as a precursor polypeptide, HA0, which is posttranslationally cleaved to two polypeptides, HA1 and HA2, linked by a disulfide bond. MAbs against the HA1 glycopolypeptide (gp) are known to neutralize the infectivity of the virus and hence provide good protection against infection (12). However, they are less efficient against heterologous or mutant strains, which are continuously arising due to antigenic shift and, to an extent, drift. Recent strategies for alternative therapy explore the more conserved epitopes of the influenza virus antigens (18, 33), which not only have the potential to stimulate a protective immune response but are also conserved among different subtypes, so as to offer protection against a broader range of viruses.The HA2 polypeptide represents a highly conserved region of HA across influenza A virus strains. The HA2 gp is responsible for the fusion of the virus and the host endosomal membrane during the entry of the virus into the cell (16). Previously, anti-HA MAbs that lacked HA inhibition activity were studied and were found to reduce the infectivity of non-H5 influenza virus subtypes by inhibition of fusion during viral replication (14). They are known to block fusion of the virus to the cell membrane at the postbinding and prefusion stage, thereby inhibiting viral replication. Furthermore, in vivo studies show that anti-HA2 MAbs that exhibit fusion inhibition activity contribute to protection and recovery from H3N2 influenza A virus infection (8). It is interesting that although the HA2 gp is generally conserved, the fusion peptide represents the most conserved region of the HA protein. So far, there have been no studies on the possible therapeutic effects of MAbs, specifically against the fusion peptide of HA, on lethal HPAI H5N1 infections.Previous studies have suggested that HA2 could contain a potential epitope responsible for the induction of antibody-mediated protective immunity (9). In the present study, a panel of MAbs against HA2 gp was characterized for their respective epitopes by epitope mapping. The therapeutic and prophylactic efficacies of these MAbs were evaluated in mice challenged with HPAI H5N1 virus infection.     

Combination Therapy Using Chimeric Monoclonal Antibodies Protects Mice from Lethal H5N1 Infection and Prevents Formation of Escape Mutants

Background

Given that there is a possibility of a human H5N1 pandemic and the fact that the recent H5N1 viruses are resistant to the anti-viral drugs, newer strategies for effective therapy are warranted. Previous studies show that single mAbs in immune prophylaxis can be protective against H5N1 infection. But a single mAb may not be effective in neutralization of a broad range of different strains of H5N1 and control of potential neutralization escape mutants.

Methods/Principal Findings

We selected two mAbs which recognized different epitopes on the hemagglutinin molecule. These two mAbs could each neutralize in vitro escape mutants to the other and in combination could effectively neutralize viruses from clades 0, 1, 2.1, 2.2, 2.3, 4, 7 and 8 of influenza A H5N1 viruses. This combination of chimeric mAbs when administered passively, pre or post challenge with 10 MLD50 (50% mouse lethal dose) HPAI H5N1 influenza A viruses could protect 100% of the mice from two different clades of viruses (clades 1 and 2.1). We also tested the efficacy of a single dose of the combination of mAbs versus two doses. Two doses of the combination therapy not only affected early clearance of the virus from the lung but could completely prevent lung pathology of the H5N1 infected mice. No escape variants were detected after therapy.

Conclusions/Significance

Our studies provide proof of concept that the synergistic action of two or more mAbs in combination is required for preventing the generation of escape mutants and also to enhance the therapeutic efficacy of passive therapy against H5N1 infection. Combination therapy may allow for a lower dose of antibody to be administered for passive therapy of influenza infection and hence can be made available at reduced economic costs during an outbreak.     

Marker-assisted improvement of bacterial blight resistance in parental lines of Pusa RH10, a superfine grain aromatic rice hybrid

Pusa RH10, the widely cultivated superfine grain aromatic rice hybrid, and its parental lines Pusa6B and PRR78 are susceptible to bacterial blight (BB) disease caused by Xanthomonas oryzae pv. oryzae. Pusa1460, a Basmati rice variety, was utilized as the donor for introgressing BB resistance genes xa13 and Xa21 into Pusa6B and PRR78 using a marker-assisted backcross breeding program. The markers RG136 and pTA248 linked to BB resistance genes xa13 and Xa21, respectively, were used for foreground selection. Seventy-four STMS markers polymorphic between Pusa6B and Pusa1460, and 54 STMS markers polymorphic between PRR78 and Pusa1460, were utilized for background selection to recover the recurrent parent genome ranging from 85.14 to 97.30% and 87.04 to 92.81% in the 10 best BC₂F₅ families of Pusa6B and PRR78, respectively. RM6100, an STMS marker linked to fertility restorer gene (Rf), was used for marker-assisted selection of Rf gene in an improved version of PRR78. The extent of donor segments in the improved version of Pusa6B was estimated to be <0.97 and <2.15 Mb in the genomic regions flanking xa13 and Xa21, respectively, whereas in improved PRR78, it was estimated to be <2.07 and <3.45 Mb in the corresponding genomic regions. Improved lines of Pusa6B and PRR78 showed yield advantages of up to 8.24 and 5.23%, respectively. The performance of the BB-resistant version of Pusa RH10 produced by intercrossing the improved parental lines was on a par with or superior to the original Pusa RH10.     

Protein engineering for covalent immobilization and enhanced stability through incorporation of multiple noncanonical amino acids

In this study, we demonstrate the application of multiple functional properties of proteins generated through coupling of residue-specific and site-specific incorporation method. With green fluorescent protein (GFP) as a model protein, we constructed multifunctional GFP through sitespecific incorporation of L-3,4-dihydroxyphenylalanine (DOPA) and residue-specific incorporation of (2S, 4S)-4- fluoroproline (4S-FP) or L-homopropargylglycine (hpg). Fluorescence analysis revealed a conjugation efficiency of approximately 20% for conjugation of DOPA-containing variants GFPdopa, GFPdp[4S-FP], and GFPdphpg onto chitosan. While incorporation of 4S-FP improved protein folding and stability, hpg incorporation into GFP allowed conjugation with fluorescent dye/polyethylene glycol (PEG). In addition, the modification of GFPhpg and GFPdphpg with PEG through Cu(I)-catalyzed click reaction increased protein thermal stability by about two-fold of the wild-type GFP.     

SNP discovery from next-generation transcriptome sequencing data and their validation using KASP assay in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Single nucleotide polymorphisms (SNPs) are becoming the most amenable form of DNA-based molecular markers for genetic analysis. In hexaploid bread wheat (Triticum aestivum L.), it is difficult to discern true polymorphic SNPs due to homoeologous and paralogous genes. Two serial analysis of gene expression (SAGE) libraries were developed utilizing leaves from resistant plants carrying leaf rust resistance gene Lr28; one library was derived from leaves that were mock inoculated and the other was derived from leaves inoculated with the urediniospores of the leaf rust pathogen Puccinia triticina. Next-generation sequencing reads, after quality trimming and removal of fungal sequences, were mapped to wheat reference sequences at Ensembl Plants. CLC Genomics Workbench and Freebayes softwares were employed for SNP calling. A total of 611 SNPs were predicted to be common by both softwares, of which 207 varietal SNPs were identified by ConservedPrimer software. A subset of 100 SNPs was used for validation across 47 wheat genotypes using Kompetitive Allele Specific PCR (KASP) assay; 83 SNPs could be successfully validated. These SNPs were positioned on wheat subgenomes and chromosome arms. When functionally annotated, many sequences harboring SNPs showed homology to resistance and resistance-like genes listed in Plant Resistance Gene database (PRGdb) as well as pathogenesis-related (PR) and stress-responsive genes. The results of the present study involving discovery of SNPs associated with resistance to leaf rust, a major threat to wheat production worldwide, will be valuable for molecular breeding for rust resistance.     

Advances in plant growth-promoting bacterial inoculant technology: formulations and practical perspectives (1998–2013)

Background

Inoculation of plants to enhance yield of crops and performance of other plants is a century old, proven technology for rhizobia and a newer venue for plant growth-promoting bacteria and other plant symbionts. The two main aspects dominating the success of inoculation are the effectiveness of the bacterial isolate and the proper application technology.

Scope

An assessment of practical aspects of bacterial inoculants for contemporary agriculture and environmental restoration is critically evaluated from the point of view of their current technological status, current applications, and future use. This is done because there are windows of opportunity for new developments in applied research using renewable, non-contaminated natural resources and new venues for research. Special emphasis is given to formulations and polymeric carriers. This review concentrates on practical aspect of inoculation technology dating from 1998 to 2013. Earlier publications are mentioned only for clarification of a specific point.

Conclusions

This review discusses characteristics of a carrier for inoculants, formulations of inoculants including liquid, organic, inorganic, polymeric, and encapsulated formulations. Technical aspects include inoculation techniques (soil and seed application), mass culture production, bulk sterilization, seed coating, shelf-life, and effect of moisture. Future research venues needed are noted.     

A unique immuno-stimulant steroidal sapogenin acid from the roots of Asparagus racemosus

A new steroidal sapogenin molecule 1 having unique characteristics, 21-nor and unusual C19 carboxylic acid has been isolated from the roots of Asparagus racemosus. On the basis of chemical evidence, extensive spectroscopic analysis including two dimensional (2D) NMR and X-ray studies of single crystal, the structure of 1 was determined as (1S,2R,3S,8S,9S,10S,13S,14S,16S,17R,22R,25R)-21-nor-18β,27α-dimethyl-1β,2β,3β-trihydroxy-25-spirost-4-en-19β-oic acid. 1 crystallizes in monoclinic space group P2₁ with a = 9.295(2), b = 11.238(2), c = 11.376(2) Å; β = 91.993(4)°, Z = 2, D_cal = 1.344 Mg/m³. The structure was solved by direct methods and refined by full-matrix least-squares procedure to a final R-value of 0.0561 for 4064 observed reflections. 1 was tested against the type of immune responses generated during treatment in normal and immune-suppressed animals and detailed biological activity evaluation suggests it to be a potent immunostimulator.     

Enhancing effects of 24-epibrassinolide and Putrescine on the antioxidant capacity and free radical scavenging activity of <Emphasis Type="Italic">Raphanus sativus</Emphasis> seedlings under Cu ion stress

The present investigation recorded significant restoration of seedling growth (root length, shoot length and fresh weight) upon application of 24-epibrassinolide (EBL) and putrescine (Put) to 7-day-old seedlings of Raphanus sativus L. cv. Pusa chetki grown under copper (Cu) ion stress. EBL and Put with/or without Cu ion treated seedlings showed increased titers of ascorbic acid, total phenols and proline when compared with Cu-stressed seedlings. Differential responses in the activities of guaiacol peroxidase (GPOX) and catalase (CAT) were noted for EBL and Put alone or with/or without Cu ion treatment. Decreased activities of glutathione reductase (GR) and superoxide dismutase (SOD) noted for EBL and Put alone were observed to enhance significantly when applied in combination with Cu ion solution. A remarkable decrease in malondialdehyde contents was observed in seedlings treated with EBL and Put alone and with/or without Cu ion stress. Enhanced free radical scavenging activities were also recorded for seedlings given EBL and Put alone or in combination over Cu ion stressed seedlings. Maximum DPPH activity was observed in seedlings treated with Put and EBL 10⁻⁹ M + Put. Significant enhancements in deoxyribose and reducing power activities were too recorded for Put, EBL and Put + 10⁻⁹ M EBL treatments. Improved seedling growth, antioxidant levels (ascorbic acid, total phenols and proline) and enzymic (GPOX, CAT, SOD and GR) activities and free radical scavenging capacities along with reduced membrane damage in seedlings given EBL and Put with/or without Cu stress suggests significant and positive interactions of EBL and Put in alleviating the Cu ion induced oxidative damage in radish seedlings.