Pigeonpea genomics initiative (PGI): an international effort to improve crop productivity of pigeonpea (Cajanus cajan L.).

Pigeonpea (Cajanus cajan), an important food legume crop in the semi-arid regions of the world and the second most important pulse crop in India, has an average crop productivity of 780 kg/ha. The relatively low crop yields may be attributed to non-availability of improved cultivars, poor crop husbandry and exposure to a number of biotic and abiotic stresses in pigeonpea growing regions. Narrow genetic diversity in cultivated germplasm has further hampered the effective utilization of conventional breeding as well as development and utilization of genomic tools, resulting in pigeonpea being often referred to as an 'orphan crop legume'. To enable genomics-assisted breeding in this crop, the pigeonpea genomics initiative (PGI) was initiated in late 2006 with funding from Indian Council of Agricultural Research under the umbrella of Indo-US agricultural knowledge initiative, which was further expanded with financial support from the US National Science Foundation's Plant Genome Research Program and the Generation Challenge Program. As a result of the PGI, the last 3 years have witnessed significant progress in development of both genetic as well as genomic resources in this crop through effective collaborations and coordination of genomics activities across several institutes and countries. For instance, 25 mapping populations segregating for a number of biotic and abiotic stresses have been developed or are under development. An 11X-genome coverage bacterial artificial chromosome (BAC) library comprising of 69,120 clones have been developed of which 50,000 clones were end sequenced to generate 87,590 BAC-end sequences (BESs). About 10,000 expressed sequence tags (ESTs) from Sanger sequencing and ca. 2 million short ESTs by 454/FLX sequencing have been generated. A variety of molecular markers have been developed from BESs, microsatellite or simple sequence repeat (SSR)-enriched libraries and mining of ESTs and genomic amplicon sequencing. Of about 21,000 SSRs identified, 6,698 SSRs are under analysis along with 670 orthologous genes using a GoldenGate SNP (single nucleotide polymorphism) genotyping platform, with large scale SNP discovery using Solexa, a next generation sequencing technology, is in progress. Similarly a diversity array technology array comprising of ca. 15,000 features has been developed. In addition, >600 unique nucleotide binding site (NBS) domain containing members of the NBS-leucine rich repeat disease resistance homologs were cloned in pigeonpea; 960 BACs containing these sequences were identified by filter hybridization, BES physical maps developed using high information content fingerprinting. To enrich the genomic resources further, sequenced soybean genome is being analyzed to establish the anchor points between pigeonpea and soybean genomes. In addition, Solexa sequencing is being used to explore the feasibility of generating whole genome sequence. In summary, the collaborative efforts of several research groups under the umbrella of PGI are making significant progress in improving molecular tools in pigeonpea and should significantly benefit pigeonpea genetics and breeding. As these efforts come to fruition, and expanded (depending on funding), pigeonpea would move from an 'orphan legume crop' to one where genomics-assisted breeding approaches for a sustainable crop improvement are routine.

Medicago truncatula EST-SSRs reveal cross-species genetic markers for Medicago spp.

Expressed sequence tags (ESTs) are important resources for gene discovery and molecular marker development. From over 147,000 ESTs of Medicago truncatula, we have identified 4,384 ESTs containing perfect simple sequence repeats (EST-SSR) of di-, tri-, tetra- or pentanucleotides. Six hundred sixteen primer pairs (PPs) were designed and screened over a panel of eight genotypes representing six Medicago spp. and subspecies. Nearly, 74% (455) of the PPs produced characteristic SSR bands of expected size length in at least one Medicago species. Four hundred six (89%) of these 455 PPs produced SSR bands in all eight genotypes tested. Only 17 PPs were M. truncatula -specific. High levels of polymorphism (>70%) were detected for these markers in alfalfa, M. truncatula, and other annual medics. About 48% of the reported markers are part of gene transcripts linked to putative functions. Our results indicate that the SSR markers developed from M. truncatula ESTs are valuable genetic markers for the Medicago genus. These markers will be useful in establishing the genomic relationships of M. truncatula to important forage legume crops such as alfalfa and other annual medics.

Chloroplast lysates support directed mutagenesis via modified DNA and chimeric RNA/DNA oligonucleotides.

Chimeric RNA/DNA and modified DNA oligonucleotides have been shown to direct gene-conversion events in vitro through a process involving proteins from several DNA-repair pathways. Recent experiments have extended the utility of these molecules to plants, and we previously demonstrated that plant cell-free extracts are competent to support oligonucleotide-directed genetic repair. Using this system, we are studying Arabidopsis DNA-repair mutants and the role of plant proteins in the DNA-repair process. Here we describe a method for investigating mechanisms of plastid DNA-repair pathways. Using a genetic readout system in bacteria and chimeric or modified DNA oligonucleotides designed to direct the conversion of mutations in antibiotic resistance genes, we have developed an assay for genetic repair of mutations in a spinach chloroplast lysate system. We report genetic repair of point and frameshift mutations directed by both types of modified oligonucleotides. This system enables the mechanistic study of plastid gene repair and facilitates the direct comparison between plant nuclear and organelle DNA-repair pathways.

Development of a transformation system for Mycosphaerella pathogens of banana: a tool for the study of host/pathogen interactions.

A genetic transformation system has been developed for three Mycosphaerella pathogens of banana and plantain (Musa spp.). Mycosphaerella fijiensis and Mycosphaerella musicola, the causal agents of black and yellow Sigatoka, respectively, and Mycosphaerella eumusae, which causes Septoria leaf spot of banana, were transformed with a construct carrying a synthetic gene encoding green fluorescent protein (GFP). Most single-spored transformants that expressed GFP constitutively were mitotically stable in the absence of selection for hygromycin B resistance. Transformants of all three species were pathogenic on the susceptible banana cultivar Grand Nain, and growth in planta was comparable to wild-type strains. GFP expression by transformants allowed us to observe extensive fungal growth within leaf tissue that eventually turned necrotic, at which point the fungi grew saprophytically on the dead tissue. Leaf chlorosis and necrosis were often observed in advance of saprophytic growth of the mycelium on necrotic tissue, which supports previous reports suggesting secretion of a phytotoxin.

The Medicago Genome Initiative: a model legume database.

The Medicago Genome Initiative (MGI) is a database of EST sequences of the model legume MEDICAGO: truncatula. The database is available to the public and has resulted from a collaborative research effort between the Samuel Roberts Noble Foundation and the National Center for Genome Resources to investigate the genome of M.truncatula. MGI is part of the greater integrated MEDICAGO: functional genomics program at the Noble Foundation (http://www.noble.org ), which is taking a global approach in studying the genetic and biochemical events associated with the growth, development and environmental interactions of this model legume. Our approach will include: large-scale EST sequencing, gene expression profiling, the generation of M.truncatula activation-tagged and promoter trap insertion mutants, high-throughput metabolic profiling, and proteome studies. These multidisciplinary information pools will be interfaced with one another to provide scientists with an integrated, holistic set of tools to address fundamental questions pertaining to legume biology. The public interface to the MGI database can be accessed at http://www.ncgr.org/research/mgi.

Agrobacterium -mediated transformation of embryogenic cell suspensions of the banana cultivar Rasthali (AAB).

A protocol was developed for establishing embryogenic suspension cultures from in vitro-grown, thin shoot-tip sections of the banana cultivar Rasthali. The best medium for callus induction was an MS-based medium supplemented with 2 mg/l 2,4-D and 0.2 mg/l zeatin. The callus was transferred to liquid medium to establish embryogenic cell suspensions. These cultures were subsequently used for Agrobacterium-mediated transformation. The Agrobacterium tumefaciens strain EHA105 containing the binary vector pVGSUN with the als gene as a selectable marker and an intron-containing the gusA gene as a reporter gene was used for transformations. The herbicide Glean was used as a selection agent. Two hundred putative transformants were recovered, of which a set of 16 was tested by histochemical analysis for GUS expression and by Southern blot analysis with a probe for the gusA gene. The plants were positive for GUS expression and integration of the gusA gene. Two of the transformants were grown to maturity under greenhouse conditions. Bananas were harvested to test GUS expression by histochemical analysis. The fruit from both transgenics tested positive for GUS expression.

Fruit-specific lectins from banana and plantain.

One of the predominant proteins in the pulp of ripe bananas (Musa acuminata L.) and plantains (Musa spp.) has been identified as a lectin. The banana and plantain agglutinins (called BanLec and PlanLec, respectively) were purified in reasonable quantities using a novel isolation procedure, which prevented adsorption of the lectins onto insoluble endogenous polysaccharides. Both BanLec and PlanLec are dimeric proteins composed of two identical subunits of 15 kDa. They readily agglutinate rabbit erythrocytes and exhibit specificity towards mannose. Molecular cloning revealed that BanLec has sequence similarity to previously described lectins of the family of jacalin-related lectins, and according to molecular modelling studies has the same overall fold and three-dimensional structure. The identification of BanLec and PlanLec demonstrates the occurrence of jacalin-related lectins in monocot species, suggesting that these lectins are more widespread among higher plants than is actually believed. The banana and plantain lectins are also the first documented examples of jacalin-related lectins, which are abundantly present in the pulp of mature fruits but are apparently absent from other tissues. However, after treatment of intact plants with methyl jasmonate, BanLec is also clearly induced in leaves. The banana lectin is a powerful murine T-cell mitogen. The relevance of the mitogenicity of the banana lectin is discussed in terms of both the physiological role of the lectin and the impact on food safety.

Identification and chromosomal localization of the monkey retrotransposon in Musa sp.

Retroelements are ubiquitous features of eukaryotic genomes, often accounting for a substantial fraction of their total DNA content. One major group of retroelements, which includes the gypsy and copia-like elements, is distinguished by the presence of long terminal repeats (LTRs). We have identified and partially characterized a sequence from banana (Musa acuminata cv. Grand Nain) which shows significant homology to gypsy-like LTR retroelements from other species. The element, named monkey, shows a high degree of homology to the reverse transcriptase, RNase H and integrase genes of retroelements from plants, fungi and yeast. However, several stop codons are present in the major ORF of this element, suggesting that this copy of monkey, if functional, is non-autonomous. Southern analysis indicated that monkey is present in both the A and B genomes of Musa, and that it is found in 200-500 copies per haploid genome in cv. Grand Nain. Chromosomal localization by fluorescent in-situ hybridization indicates that copies of monkey are concentrated in the nucleolar organizer regions and colocalize with rRNA genes. Other copies of monkey appear to be dispersed throughout the genome.

Genetic repair of mutations in plant cell-free extracts directed by specific chimeric oligonucleotides.

Chimeric oligonucleotides are synthetic molecules comprised of RNA and DNA bases assembled in a double hairpin conformation. These molecules have been shown to direct gene conversion events in mammalian cells and animals through a process involving at least one protein from the DNA mismatch repair pathway. The mechanism of action for gene repair in mammalian cells has been partially elucidated through the use of a cell-free extract system. Recent experiments have expanded the utility of chimeric oligonucleotides to plants and have demonstrated genotypic and phenotypic conversion, as well as Mendelian transmission. Although these experiments showed correction of point and frameshift mutations, the biochemical and mechanistic aspects of the process were not addressed. In this paper, we describe the establishment of cell-free extract systems from maize (Zea mays), banana (Musa acuminata cv Rasthali), and tobacco (Nicotiana tabacum). Using a genetic readout system in bacteria and chimeric oligonucleotides designed to direct the conversion of mutations in antibiotic-resistant genes, we demonstrate gene repair of point and frameshift mutations. Whereas extracts from banana and maize catalyzed repair of mutations in a precise fashion, cell-free extracts prepared from tobacco exhibited either partial repair or non-targeted nucleotide conversion. In addition, an all-DNA hairpin molecule also mediated repair albeit in an imprecise fashion in all cell-free extracts tested. This system enables the mechanistic study of gene repair in plants and may facilitate the identification of DNA repair proteins operating in plant cells.

Purification, characterization and structural analysis of an abundant beta-1,3-glucanase from banana fruit.

An abundant, catalytically active beta-1,3-endoglucanase (EC 3.2.1. 39) has been isolated from the pulp of ripe bananas. Biochemical analysis of the purified protein, molecular modelling, and molecular cloning of the corresponding gene indicate that this banana enzyme closely resembles previously characterized plant beta-glucanases with respect to its amino-acid sequence, structure and biological activity. The results described in this paper demonstrate both the occurrence of an abundant active beta-1,3-endoglucanases in fruits and also readdress the question of the possible involvement of these enzymes in the ripening and/or softening process.

Purification and structural analysis of an abundant thaumatin-like protein from ripe banana fruit.

The pulp of ripe bananas (Musa acuminata) contains an abundant thaumatin-like protein (TLP). Characterization of the protein and molecular cloning of the corresponding gene from banana demonstrated that the native protein consists of a single polypeptide chain of 200 amino acid residues. Molecular modelling further revealed that the banana thaumatin-like protein (Ban-TLP) adopts an overall fold similar to that of thaumatin and thaumatin-like PR-5 proteins. Although the banana protein exhibits an electrostatically polarized surface, which is believed to be essential for the antifungal properties of TLPs, it is apparently devoid of antifungal activity towards pathogenic fungi. It exhibits a low but detectable in vitro endo-beta-1,3-glucanase (EC 3.2.1.x) activity. As well as being present in fruits, Ban-TLP also occurs in root tips where its accumulation is enhanced by methyl jasmonate treatment of plants. Pulp of plantains (Musa acuminata) also contains a very similar TLP, which is even more abundant than its banana homologue. Our results demonstrate for the first time that fruit-specific (abundant) TLPs are not confined to dicots but occur also in fruits of monocot species. The possible role of the apparent widespread accumulation of fruit-specific TLPs is discussed.

A tool for functional plant genomics: chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations.

Self-complementary chimeric oligonucleotides (COs) composed of DNA and modified RNA residues were evaluated as a means to (i) create stable, site-specific base substitutions in a nuclear gene and (ii) introduce a frameshift in a nuclear transgene in plant cells. To demonstrate the creation of allele-specific mutations in a member of a gene family, COs were designed to target the codon for Pro-196 of SuRA, a tobacco acetolactate synthase (ALS) gene. An amino acid substitution at Pro-196 of ALS confers a herbicide-resistance phenotype that can be used as a selectable marker in plant cells. COs were designed to contain a 25-nt homology domain comprised of a five-deoxyribonucleotide region (harboring a single base mismatch to the native ALS sequence) flanked by regions each composed of 10 ribonucleotides. After recovery of herbicide-resistant tobacco cells on selective medium, DNA sequence analyses identified base conversions in the ALS gene at the codon for Pro-196. To demonstrate a site-specific insertion of a single base into a targeted gene, COs were used to restore expression of an inactive green fluorescent protein transgene that had been designed to contain a single base deletion. Recovery of fluorescent cells confirmed the deletion correction. Our results demonstrate the application of a technology to modify individual genetic loci by catalyzing either a base substitution or a base addition to specific nuclear genes; this approach should have great utility in the area of plant functional genomics.

The abundant 31-kilodalton banana pulp protein is homologous to class-III acidic chitinases.

We have identified and characterized the abundant protein from the pulp of banana fruit (Musa acuminata cv. Grand Nain), and have isolated a cDNA clone encoding this protein. Comparison of the amino terminal sequence of the purified 31 kDa protein (P31) suggests that it is related to plant chitinases. Western analyses utilizing rabbit anti-P31 antiserum demonstrate that this protein is pulp-specific in banana. A full-length cDNA clone homologous to class III acidic chitinase genes has been isolated from a pulp cDNA library by differential screening. The identity of this clone as encoding P31 was verified by comparisons between the amino-terminal peptide sequence and the cDNA sequence and cross-hybridization of the translation product of the cDNA clone with P31 antiserum. Northern and western blot analyses of RNA and protein isolated from banana pulp at different stages of ripening indicate that the cDNA and protein are expressed at high levels in the pulp of unripe fruit, and that their abundance decreases as the fruit ripens. Based on its expression pattern and deduced amino acid sequence and composition, we hypothesize that the physiological role of P31 is not for plant protection, but as a storage protein in banana pulp.

Differential gene expression in ripening banana fruit.

During banana (Musa acuminata L.) fruit ripening ethylene production triggers a developmental cascade that is accompanied by a massive conversion of starch to sugars, an associated burst of respiratory activity, and an increase in protein synthesis. Differential screening of cDNA libraries representing banana pulp at ripening stages 1 and 3 has led to the isolation of 11 nonredundant groups of differentially expressed mRNAs. Identification of these transcripts by partial sequence analysis indicates that two of the mRNAs encode proteins involved in carbohydrate metabolism, whereas others encode proteins thought to be associated with pathogenesis, senescence, or stress responses in plants. Their relative abundance in the pulp and tissue-specific distribution in greenhouse-grown banana plants were determined by northern-blot analyses. The relative abundance of transcripts encoding starch synthase, granule-bound starch synthase, chitinase, lectin, and a type-2 metallothionein decreased in pulp during ripening. Transcripts encoding endochitinase, beta-1,3-glucanase, a thaumatin-like protein, ascorbate peroxidase, metallothionein, and a putative senescence-related protein increased early in ripening. The elucidation of the molecular events associated with banana ripening will facilitate a better understanding and control of these processes, and will allow us to attain our long-term goal of producing candidate oral vaccines in transgenic banana plants.

Nuclear protein factors binding to a class I patatin promoter region are tuber-specific and sucrose-inducible.

Genes encoding patatin, the major storage protein of the potato tuber, are generally divided into two classes, class I and class II. The expression of the class I patatin genes is normally tuber-specific, but can be induced in leaves by high concentrations of sucrose. By employing electrophoretic mobility shift assays (EMSA), we have identified nuclear protein factors that interact specifically with the proximal portion of the class I patatin promoter that is required for tuber-specific and sucrose-inducible expression. The factors were detected in nuclear extracts prepared from potato tubers and sucrose-induced leaves, but not in extracts from leaves of normal potato plants. Four putative transcription factor-binding sites were localized using DNase I footprinting. Competitive EMSA was employed to show that the same protein factor binds to at least two of the sites (boxes D and M). Interestingly, these two binding sites are highly homologous to light-responsive elements present in genes for the ribulose-1,5-bisphosphate carboxylase small subunit.

Phosphate Modulates Transcription of Soybean VspB and Other Sugar-Inducible Genes.

The soybean vegetative storage protein genes VspA and VspB encode vacuolar glycoprotein acid phosphatases. Transcription of the Vsp is synergistically activated by jasmonic acid or methyl jasmonate (MeJA) and soluble sugars. The action of these modulators is mediated by two different DNA domains in the VspB promoter. In this study, we present new data regarding VspB regulation by sucrose and inorganic phosphate, which suggest a common mechanism of transcriptional control for Vsp and other sugar-inducible genes. We found that the sugar-mediated activation of VspB expression was inhibited by phosphate. Deletion analysis and transient assays in tobacco protoplasts identified a 130-bp DNA domain in the VspB promoter that mediates both sucrose induction and phosphate inhibition. Transcription mediated by this DNA domain was induced by phosphate elimination from the protoplast incubation medium, even in the absence of sucrose. The effect of sucrose and phosphate on VspB expression was studied in vivo in several ways. Depletion of phosphate from soybean cell cultures by the addition of mannose stimulated VspB expression, even in the absence of sucrose or MeJA. In illuminated soybean leaves treated with MeJA, inhibition of photosynthetic electron transport by DCMU decreased VspB expression. In contrast, VspB expression in soybean leaves stimulated by phosphate depletion was not influenced by DCMU. Moreover, sucrose-stimulated expression of the sugar-responsive genes lipoxygenase A and chalcone synthase of soybean and proteinase inhibitor II and class I patatin of potato was inhibited by phosphate. Like VspB, these genes were stimulated by phosphate depletion in the absence of exogenous sucrose. We propose that sugar-responsive genes are activated, in part, by accumulation of sugar-phosphates and concomitant reduction of cellular phosphate levels. These data may help explain recruitment of the Vsp, which encode acid phosphatases, as vegetative storage proteins.