We present a combined computational and experimental method for the rapid optimization of proteins. Using ␤-lactamase as a test case, we redesigned the active site region using our Protein Design Automation technology as a computational screen to search the entire sequence space. By eliminating sequences incompatible with the protein fold, Protein Design Automation rapidly reduced the number of sequences to a size amenable to experimental screening, resulting in a library of Ϸ200,000 mutants. These were then constructed and experimentally screened to select for variants with improved resistance to the antibiotic cefotaxime. In a single round, we obtained variants exhibiting a 1,280-fold increase in resistance. To our knowledge, all of the mutations were novel, i.e., they have not been identified as beneficial by random mutagenesis or DNA shuffling or seen in any of the naturally occurring TEM ␤-lactamases, the most prevalent type of Gram-negative ␤-lactamases. This combined approach allows for the rapid improvement of any property that can be screened experimentally and provides a powerful broadly applicable tool for protein engineering.computational protein design ͉ protein engineering ͉ mutagenesis ͉ directed evolution ͉ ␤-lactamase

Expression of ACT, EF1A; H2A, EF1A, ACT and 18S, TUB showed stability under MYMIV, salinity and drought stress, respectively; these are recommended as reference genes for qPCR normalization in Vigna mungo. Accurate gene expression profiling through qPCR depends on selection of appropriate reference gene(s) for normalization. Due to lack of unanimous internal standard, suitable constitutively expressed reference genes are selected that exhibit stable expression under diverse experimental conditions. In this communication, a comparative evaluation of stability among seven V. mungo genes encoding actin (ACT), histone H2A (H2A), elongation factor 1-alpha (EF1A), 18S rRNA (18S), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cyclophilin (CYP) and tubulin (TUB) under biotic (MYMIV) and abiotic (drought and salinity) stress conditions has been attempted. Specificity and amplification efficiency for each primer pair were verified; however, cumulative assessment of their accumulated transcripts revealed no uniformity. Therefore, individual stability and suitability of these seven candidates have been assessed in silico, by two widely used algorithms, geNorm and Normfinder. Based on the computed results, high stability was obtained for ACT and EF1A during MYMIV stress, while H2A, EFIA and ACT were found to be most suitable in salinity stress experiments and TUB and 18S during drought treatments. Combinations of ACT/TUB or ACT/EFIA were recommended for their use in the pooled analysis, while expression of 18S and CYP showed greater variations and therefore considered unsuitable as reference genes. Additionally, precise quantification of the target gene VmPRX under these stresses was shown to be a function of reference genes' stability, which tends to get affected when normalized with the least stable genes. Hence, use of these normalizers will facilitate accurate and reliable analyses of gene expression in V. mungo.

BackgroundVigna mungo, a tropical leguminous plant, highly susceptible to yellow mosaic disease caused by Mungbean Yellow Mosaic India Virus (MYMIV) resulting in high yield penalty. The molecular events occurring during compatible and incompatible interactions between V. mungo and MYMIV pathosystem are yet to be explored. In this study biochemical analyses in conjunction with proteomics of MYMIV-susceptible and -resistant V. mungo genotypes were executed to get an insight in the molecular events during compatible and incompatible plant-virus interactions.ResultsBiochemical analysis revealed an increase in phenolics, hydrogen peroxide and carbohydrate contents in both compatible and incompatible interactions; but the magnitudes were higher during incompatible interaction. In the resistant genotype the activities of superoxide dismutase and ascorbate peroxidase increased significantly, while catalase activity decreased. Comparative proteome analyses using two-dimensional gel electrophoresis coupled with mass spectrometry identified 109 differentially abundant proteins at 3, 7 and 14 days post MYMIV-inoculation. Proteins of several functional categories were differentially changed in abundance during both compatible and incompatible interactions. Among these, photosynthesis related proteins were mostly affected in the susceptible genotype resulting in reduced photosynthesis rate under MYMIV-stress. Differential intensities of chlorophyll fluorescence and chlorophyll contents are in congruence with proteomics data. It was revealed that Photosystem II electron transports are the primary targets of MYMIV during pathogenesis. Quantitative real time PCR analyses of selected genes corroborates with respective protein abundance during incompatible interaction. The network of various cellular pathways that are involved in inducing defense response contains several conglomerated cores of nodal proteins, of which ascorbate peroxidase, rubisco activase and serine/glycine hydroxymethyl transferase are the three major hubs with high connectivity. These nodal proteins play the crucial role of key regulators in bringing about a coordinated defense response in highly orchestrated manner.ConclusionsBiochemical and proteomic analyses revealed early accumulation of the defense/stress related proteins involved in ROS metabolism during incompatible interaction. The robustness in induction of defense/stress and signal transduction related proteins is the key factor in inducing resistance. The mechanism of MYMIV-resistance in V. mungo involves redirection of carbohydrate flux towards pentose phosphate pathway. Some of these identified, differentially regulated proteins are also conferring abiotic stress responses illustrating harmony amongst different stress responses. To the best of our knowledge, this is the lone study deciphering differential regulations of V. mungo leaf proteome upon MYMIV infection elucidating the mode of resistance response at the biochemical level.

Mungbean Yellow Mosaic India Virus (MYMIV)-infection creates major hindrance in V . mungo cultivation and poses significant threat to other grain legume production. Symptoms associated include severe patho-physiological alterations characterized by chlorotic foliar lesion accompanied by reduced growth. However, dissection of the host’s defense machinery remains a tough challenge due to limited of host’s genomic resources. A comparative RNA-Seq transcriptomes of resistant (VM84) and susceptible (T9) plants was carried out to identify genes potentially involved in V . mungo resistance against MYMIV. Distinct gene expression landscapes were observed in VM84 and T9 with 2158 and 1679 differentially expressed genes (DEGs), respectively. Transcriptomic responses in VM84 reflect a prompt and intense immune reaction demonstrating an efficient pathogen surveillance leading to activation of basal and induced immune responses. Functional analysis of the altered DEGs identified multiple regulatory pathways to be activated or repressed over time. Up-regulation of DEGs including NB-LRR, WRKY33, ankyrin, argonaute and NAC transcription factor revealed an insight on their potential roles in MYMIV-resistance; and qPCR validation shows a propensity of their accumulation in VM84. Analyses of the current RNA-Seq dataset contribute immensely to decipher molecular responses that underlie MYMIV-resistance and will aid in the improvement strategy of V . mungo and other legumes through comparative functional genomics.

Mungbean Yellow Mosaic India Virus (MYMIV) belonging to the genus begomovirus causes the yellow mosaic disease in a number of economically important edible grain legumes including mungbean (Vigna radiata), urdbean (Vigna mungo) and soybean (Glycine max). The disease is severe, critical, open spread and inflicts heavy yield losses annually. The objective of this study is to develop molecular markers linked to MYMIV-resistance to facilitate genotyping of urdbean and mungbean germplasms for MYMIV-reaction. Resistance-linked molecular markers were successfully developed from consensus motifs of other resistance (R) gene or R gene homologue sequences. Applying linked marker-assisted genotyping, plant breeders can carry out repeated genotyping throughout the growing season in absence of any disease incidence. Two MYMIV-resistance marker loci, YR4 and CYR1, were identified and of these two CYR1 is completely linked with MYMIV-resistant germplasms and co-segregating with MYMIV-resistant F₂, F₃ progenies of urdbean. The present study demonstrated that these two markers could be efficiently employed together in a multiplex-PCR-reaction for genotyping both V. mungo and V. radiata germplasms from field grown plants and also directly from the seed stock. This method of genotyping would save time and labour during the introgression of MYMIV-resistance through molecular breeding, as methods of phenotyping against begomoviruses are tedious, labour and time intensive.

MicroRNAs (miRNAs) are 20-24 nucleotide long non-coding RNAs known to play important regulatory roles during plant development, organ morphogenesis, and stress responses by controlling gene expression. Although Vigna unguiculata (cowpea) is an economically important salt sensitive member of legumes, very little is known about the conserved miRNAs and their expression profile during salinity stress in this plant. In the present study using comparative genomic approach and following a set of strict filtering criteria we have identified 18 conserved V. unguiculata miRNAs belonging to 16 distinct miRNA families. Using these potential miRNA sequences 15 potential target genes were predicted and all of them were identified as transcription factors. Seven of these predicted V. unguiculata miRNAs were experimentally validated in the root tissues and found to be up-regulated during salt stress as revealed by quantitative real time PCR (qRT-PCR). Perfectly cleaved Auxin response factor (ARF), the target transcript of V. unguiculata miR160 was detected successfully by modified 5 0 RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) method.

Granulocyte-colony stimulating factor (G-CSF) is used worldwide to prevent neutropenia caused by highdose chemotherapy. It has limited stability, strict formulation and storage requirements, and because of poor oral absorption must be administered by injection (typically daily). Thus, there is significant interest in developing analogs with improved pharmacological properties. We used our ultrahigh throughput computational screening method to improve the physicochemical characteristics of G-CSF. Improving these properties can make a molecule more robust, enhance its shelf life, or make it more amenable to alternate delivery systems and formulations. It can also affect clinically important features such as pharmacokinetics. Residues in the buried core were selected for optimization to minimize changes to the surface, thereby maintaining the active site and limiting the designed protein's potential for antigenicity. Using a structure that was homology modeled from bovine G-CSF, core designs of 25-34 residues were completed, corresponding to 10 21 -10 28 sequences screened. The optimal sequence from each design was selected for biophysical characterization and experimental testing; each had 10-14 mutations. The designed proteins showed enhanced thermal stabilities of up to 13°C, displayed five-to 10-fold improvements in shelf life, and were biologically active in cell proliferation assays and in a neutropenic mouse model. Pharmacokinetic studies in monkeys showed that subcutaneous injection of the designed analogs results in greater systemic exposure, probably attributable to improved absorption from the subcutaneous compartment. These results show that our computational method can be used to develop improved pharmaceuticals and illustrate its utility as a powerful protein design tool.

Initial phases of the MYMIV- Vigna mungo interaction is crucial in determining the infection phenotype upon challenging with the virus. During incompatible interaction, the plant deploys multiple stratagems that include extensive transcriptional alterations defying the virulence factors of the pathogen. Such molecular events are not frequently addressed by genomic tools. In order to obtain a critical insight to unravel how V. mungo respond to Mungbean yellow mosaic India virus (MYMIV), we have employed the PCR based suppression subtractive hybridization technique to identify genes that exhibit altered expressions. Dynamics of 345 candidate genes are illustrated that differentially expressed either in compatible or incompatible reactions and their possible biological and cellular functions are predicted. The MYMIV-induced physiological aspects of the resistant host include reactive oxygen species generation, induction of Ca2+ mediated signaling, enhanced expression of transcripts involved in phenylpropanoid and ubiquitin-proteasomal pathways; all these together confer resistance against the invader. Elicitation of genes implicated in salicylic acid (SA) pathway suggests that immune response is under the regulation of SA signaling. A significant fraction of modulated transcripts are of unknown function indicating participation of novel candidate genes in restricting this viral pathogen. Susceptibility on the other hand, as exhibited by V. mungo Cv. T9 is perhaps due to the poor execution of these transcript modulation exhibiting remarkable repression of photosynthesis related genes resulting in chlorosis of leaves followed by penalty in crop yield. Thus, the present findings revealed an insight on the molecular warfare during host-virus interaction suggesting plausible signaling mechanisms and key biochemical pathways overriding MYMIV invasion in resistant genotype of V. mungo. In addition to inflate the existing knowledge base, the genomic resources identified in this orphan crop would be useful for integrating MYMIV-tolerance trait in susceptible cultivars of V. mungo.