Cotton microbiome profiling and Cotton Leaf Curl Disease (CLCuD) suppression through microbial consortia associated with Gossypium arboreum.

The failure of breeding strategies has caused scientists to shift to other means where the new approach involves exploring the microbiome to modulate plant defense mechanisms against Cotton Leaf Curl Disease (CLCuD). The cotton microbiome of CLCuD-resistant varieties may harbor a multitude of bacterial genera that significantly contribute to disease resistance and provide information on metabolic pathways that differ between the susceptible and resistant varieties. The current study explores the microbiome of CLCuD-susceptible Gossypium hirsutum and CLCuD-resistant Gossypium arboreum using 16 S rRNA gene amplification for the leaf endophyte, leaf epiphyte, rhizosphere, and root endophyte of the two cotton species. This revealed that Pseudomonas inhabited the rhizosphere while Bacillus was predominantly found in the phyllosphere of CLCuV-resistant G. arboreum. Using salicylic acid-producing Serratia spp. and Fictibacillus spp. isolated from CLCuD-resistant G. arboreum, and guided by our analyses, we have successfully suppressed CLCuD in the susceptible G. hirsutum through pot assays. The applied strains exhibited less than 10% CLCuD incidence as compared to control group where it was 40% at 40 days post viral inoculation. Through detailed analytics, we have successfully demonstrated that the applied microbes serve as a biocontrol agent to suppress viral disease in Cotton.

Enhanced expression of plasma membrane intrinsic protein 2 improves cotton fiber length in Gossypium arboreum.

BACKGROUND: Gossypium arboreum is a cotton crop native to tropical and subtropical regions that are naturally resistant to cotton leaf curl virus (CLCuV). However, its cultivation is unfavorable due to the lower quality and shorter fiber length of cotton when compared to the market leading G. hirsutum. Plasma membrane intrinsic protein 2 (PIP2) is an aquaporin responsible for the transport of water and small molecules across cellular membranes. This fluid transport influences cell elongation and cotton fibre development. Hence, increased PIP2 expression may yield plants with enhanced fiber qualities including length. METHODS AND RESULTS: To test this hypothesis, G. arboreum was transformed with a PIP2 gene construct (35SCpPIP2) using the Agrobacterium-mediated shoot apex cutting method. Relative expression of the CpPIP2 gene in transgenic plants increased up to 35-fold when compared with non-transgenic controls. Transgenic plants displayed a corresponding increase of staple length (up to 150%) when compared with non-transgenic controls. Transgene integration was examined using FISH and karyotyping and revealed the presence of a single transgene located on chromosome 6. CONCLUSION: Since G. arboreum is naturally whitefly and CLCuV resistant, this improvement of fiber length evidenced for CpPIP2 transgenic plants renders their crop production more economically viable.

Development of event-specific detection method for identification of insect resistant NIBGE-1601 cotton harboring double gene Cry1Ac-Cry2Ab construct.

Bt cotton expressing Cry1Ac is being cultivated in Pakistan. It has been observed that pink bollworm may have developed resistance against single Bt gene (Cry1Ac). For durable resistance, insect resistant NIBGE-1601 cotton harboring double gene Cry1Ac-Cry2Ab construct was developed. There was a need to characterize NIBGE-1601 event for intellectual property rights protection. The Presence of NIBGE Cry1Ac and NIBGE Cry2Ab genes was checked in NIBGE-1601 cotton plants through PCR, while there was no amplification using primers specific for Monsanto events (MON531, MON15985, MON1445). Using genome walking technology, NIBGE-601 event has been characterized. Event-specific primers of NIBGE-1601 were designed and evaluated to differentiate it from other cotton events mentioned above. NIBGE-1601 event detection primers are highly specific, therefore, can detect NIBGE 1601 event at different conditions using single or multiplex PCR. In the qualitative PCR, using NIBGE-1601 event specific primers, 0.05 ng was the limit of detection for NIBGE-1601double gene cotton genomic DNA. Thus event characterization and development of event-specific diagnostics will help in breeding new cotton varieties resistant to cotton bollworms.

Isolation of biotic stress resistance genes from cotton (Gossypium arboreum) and their analysis in model plant tobacco (Nicotiana tabacum) for resistance against cotton leaf curl disease complex.

Cotton production is widely effected by Cotton Leaf Curl Virus (CLCuV) in world posing serious losses to cotton yield.The CRT genes from CLCuV resistant G. arboreum and CLCuV susceptible G. hirsutum were cloned and sequenced to know the differences of protein composition in both species. Molecular techniques were used to isolate full length putative biotic stress resistance genes from G. arboreum besides the analysis of identified novel genes in model plant tobacco (Nicotiana tabacum) for resistance to cotton leaf curl disease complex. It was found that transgenic plants over expressing Hydroperoxidelyase (HPL) genes exhibited higher enzyme activity than wild type. In addition the genome sequence information was used for the purpose of gene isolation. Even for the enhanced expression of Calreticulin (CRT), AOS and HPL in G. hirsutum, it still showed susceptibility against CLCuV suggesting alternative genes and pathways involved for the expression of resistance.

Development and evaluation of double gene transgenic cotton lines expressing Cry toxins for protection against chewing insect pests.

Cotton is the main fiber producing crop globally, with a significant impact on the economy of Pakistan. Bt cotton expressing a Cry1Ac gene is grown over a large area in Pakistan, however, there is a major concern that bollworms may develop resistance. Here we have used a durable resistance strategy against bollworms by developing a double gene construct containing Cry1Ac and Cry2Ab (pGA482-12R) for cotton transformation. Both Cry toxin genes have been cloned in the same T-DNA borders and transferred successfully into cotton via Agrobacterium-mediated transformation. Both genes are expressed in transgenic cotton plants and is likely to help breeders in developing new cotton cultivars by incorporating these genes in cotton lines having no Bt genes or expressing Cry1Ac gene (Mon 531). Positive transgenic cotton was identified by PCR using specific primers for the amplification of both Cry1Ac and Cry2Ab genes. Cry1Ac and Cry2Ab expression was confirmed with an immunostrip test and quantified using ELISA that showed significant spatio-temporal expression of Cry2Ab ranging from 3.28 to 7.72 µg/g of the tissue leaf. Insect bioassay with army worm (Spodoptera litura) was performed to check the efficacy of NIBGE (National Institute for Biotechnology and Genetic Engineering) double gene transgenic cotton plants and up to 93% insect mortality was observed.

Identification of plasmid encoded osmoregulatory genes from halophilic bacteria isolated from the rhizosphere of halophytes.

Bacterial plasmids carry genes that code for additional traits such as osmoregulation, CO2 fixation, antibiotic and heavy metal resistance, root nodulation and nitrogen fixation. The main objective of the current study was to identify plasmid-conferring osmoregulatory genes in bacteria isolated from rhizospheric and non-rhizospheric soils of halophytes (Salsola stocksii and Atriplex amnicola). More than 55% of halophilic bacteria from the rhizosphere and 70% from non-rhizospheric soils were able to grow at 3 M salt concentrations. All the strains showed optimum growth at 1.5-3.0 M NaCl. Bacterial strains from the Salsola rhizosphere showed maximum (31%) plasmid elimination during curing experiments as compared to bacterial strains from the Atriplex rhizosphere and non-rhizospheric soils. Two plasmid cured strains Bacillus HL2HP6 and Oceanobacillus HL2RP7 lost their ability to grow in halophilic medium, but they grew well on LB medium. The plasmid cured strains also showed a change in sensitivity to specific antibiotics. These plasmids were isolated and transformed into E. coli strains and growth response of wild-type and transformed E. coli strains was compared at 1.5-4 M NaCl concentrations. Chromosomal DNA and plasmids from Bacillus filamentosus HL2HP6 were sequenced by using high throughput sequencing approach. Results of functional analysis of plasmid sequences showed different proteins and enzymes involved in osmoregulation of bacteria, such as trehalose, ectoine synthetase, porins, proline, alanine, inorganic ion transporters, dehydrogenases and peptidases. Our results suggested that plasmid conferring osmoregulatory genes play a vital role to maintain internal osmotic balance of bacterial cells and these genes can be used to develop salt tolerant transgenic crops.

Multiple upstream start codons (AUG) in 5' untranslated region enhance translation efficiency of cry2Ac11 without helper protein.

Cry2Ac11, a 65 kDa insecticidal protein produced by Bacillus thuringiensis, shows toxicity against dipteran and lepidopteran larvae. It is encoded by cry2Ac11 gene ( orf3), which is part of an operon comprising orf1, orf2, and orf3. Orf2, a helper protein, helps in proper folding and prevents aberrant aggregation of newly produced molecules. In this study, we have elucidated the effect of different mutations in translation initiation region (TIR), particularly the ribosomal binding site and the start codon (RBS-ATG) on cry2Ac11 gene expression without helper protein. All recombinant constructs were expressed in acrystalliferous B. thuringiensis subsp israelensis 4Q7 under the control of strong chimeric promoter cyt1AP/STAB. Of all the mutants, mut/RBS2, with two consecutive AUGs after the spacer region in TIR, exhibited 89- and 2246-fold higher transcript levels compared with 4Q7-operSalI/RBS ( cry2Ac11 operon) and 4Q7-w-RBS ( cry2Ac11 gene), respectively. The analysis of mut/RBS2 messenger RNA (mRNA) structure in the RBS-AUG region showed the presence of RBS in the single-stranded part of the moderately stable hairpin loop. The high expression efficiency of Cry2Ac11 mutant without helper protein is a cumulative and cooperative result of chimeric promoter cyt1AP/STAB-SD with the optimal context of RBS-AUG region provided by multiple AUGs and stabilizer sequence at 3' ends.

Exploration of cotton leaf curl virus resistance genes and their screening in Gossypium arboreum by targeting resistance gene analogues.

Cotton leaf curl virus (CLCuV) disease is one of the major limiting factors in cotton production, particularly in widely cultivated Gossypium hirsutum varieties that are susceptible to attack by this virus. Several approaches have been employed to explore putative resistance genes in another cotton species, G. arboreum. However, the exact mechanisms conferring disease resistance in cotton are still unknown. In the current study, we used various approaches to identify possible resistance genes against CLCuV infection. We report the identification and isolation of a set of genes involved in the resistance response to viral infestation. PCR products containing genomic DNA gave multiple amplifications with a single primer in most reactions, and 38 fragments were cloned from G. arboreum and G. hirsutum. The sequences of cloned fragments belonged to various pathway genes and uncharacterized proteins. However, five amplified fragments (RM1, RM6, RM8, RM12 and RM31) showed similarity with R genes. Maximum homology (94 %) was observed with G. raimondii toll/interleukin receptor-like protein. BLAST search showed the homology of all resistance gene analogues (RGAs) with more than one chromosome, and multiple hits were observed on each chromosome for each RGA. Expression analysis through RT-PCR identified variable expression levels of the different RGAs in all tested genotypes. The expression level of RGAs differed between symptomatic and asymptomatic plants, with the exception of RGA 395, whose expression level was the same in both diseased and healthy plants. Knowledge of the interaction of these genes with various cotton pathogens could be utilized to improve the resistance of susceptible G. hirsutum and other plant species.

Development of a Triple Gene Cry1Ac-Cry2Ab-EPSPS Construct and Its Expression in Nicotiana benthamiana for Insect Resistance and Herbicide Tolerance in Plants.

Insect pest complex, cotton leaf curl disease and weeds pose major threat to crop production worldwide, including Pakistan. To address these problems, in the present study a triple gene construct harboring Cry1Ac, Cry2Ab, and EPSPS cassettes has been developed for plant specifically in cotton transformation against lepidopteron insect-pests and weeds. Nicotiana benthamiana (tobacco) was used as a model system for characterization of this triple gene construct. The construct has been assembled in plant expression vector and transformed in N. benthamiana. In six transgenic tobacco lines the integration of Cry1Ac-Cry2Ab-EPSPS in tobacco genome was checked by PCR, while successful protein expression of all the three genes was confirmed through immunostrip assay. Efficacy of Cry1Ac and Cry2Ab was evaluated through insect bioassay using armyworm (Spodoptera littoralis). These transgenic tobacco plants showed significant insect mortality as compared to control plants during insect bioassay. Three out of six tested transgenic lines L3, L5, and L9 exhibited 100% mortality of armyworm, while three other lines L1, L10, and L7 showed 86, 80, and 40% mortality, respectively. This construct can readily be used with confidence to transform cotton and other crops for the development of insect resistant and herbicide tolerant transgenic plants. The transgenic crop plants developed using this triple gene construct will provide an excellent germplasm resource for the breeders to improve their efficiency in developing stable homozygous lines as all the three genes being in a single T-DNA border will inherit together.

Transgenic expression of phytase in wheat endosperm increases bioavailability of iron and zinc in grains.

Phytate is a major constituent of wheat seeds and chelates metal ions, thus reducing their bioavailability and so the nutritional value of grains. Transgenic plants expressing heterologous phytase are expected to enhance degradation of phytic acid stored in seeds and are proposed to increase the in vitro bioavailability of mineral nutrients. Wheat transgenic plants expressing Aspergillus japonicus phytase gene (phyA) in wheat endosperm were developed till T3 generation. The transgenic lines exhibited 18-99 % increase in phytase activity and 12-76 % reduction of phytic acid content in seeds. The minimum phytic acid content was observed in chapatti (Asian bread) as compared to flour and dough. The transcript profiling of phyA mRNA indicated twofold to ninefold higher expression as compared to non transgenic controls. There was no significant difference in grain nutrient composition of transgenic and non-transgenic seeds. In vitro bioavailability assay for iron and zinc in dough and chapatti of transgenic lines revealed a significant increase in iron and zinc contents. The development of nutritionally enhanced cereals is a step forward to combat nutrition deficiency for iron and zinc in malnourished human population, especially women and children.

Stable transformation and expression of GhEXPA8 fiber expansin gene to improve fiber length and micronaire value in cotton.

Cotton fiber is multigenic trait controlled by number of genes. Previous studies suggest that one of these genes may be responsible for switching cotton fiber growth on and off to influence the fiber quality produced from a cotton seed. In the present study, the Gossypium hirsutum GhEXPA8 fiber expansin gene was introduced into local cotton variety NIAB 846 by using an Agrobacterium-mediated gene transformation. The neomycin phosphotransferase (NPTII) gene was used as a selection marker for screening of putative transgenic cotton plants. Integration and expression of the fiber expansin gene in cotton plants was confirmed with molecular techniques including Southern blot analyses, real-time PCR. Cellulose assay was used for measurement of cellulose contents of transgenic cotton fiber. The data collected from 3 years of field performance of the transgenic cotton plants expressing GhEXPA8 showed that significant improvement has been made in fiber lengths and micronaire values as compared to control G. hirsutum variety NIAB 846 cotton plants. Statistical techniques were also used for analysis of fiber and agronomic characteristics. The results of this study support improvement of cotton fiber through genetic modification.

Identification and characterization of plasma membrane aquaporins isolated from fiber cells of Calotropis procera.

Calotropis procera, commonly known as "milkweed", possesses long seed trichomes for seed dispersal and has the ability to survive under harsh conditions such as drought and salinity. Aquaporins are water channel proteins expressed in all land plants, divided into five subfamilies plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), NOD26-like proteins (NIPs), small basic intrinsic proteins (SIPs), and the unfamiliar X intrinsic proteins (XIPs). PIPs constitute the largest group of water channel proteins that are involved in different developmental and regulatory mechanisms including water permeability, cell elongation, and stomata opening. Aquaporins are also involved in abiotic stress tolerance and cell expansion mechanisms, but their role in seed trichomes (fiber cells) has never been investigated. A large number of clones isolated from C. procera fiber cDNA library showed sequence homology to PIPs. Both expressed sequence tags (ESTs) and real-time polymerase chain reaction (PCR) studies revealed that the transcript abundance of this gene family in fiber cells of C. procera is greater than that of cotton. Full-length cDNAs of CpPIP1 and CpPIP2 were isolated from C. procera fiber cDNA library and used for constructing plant expression vectors under constitutive (2×35S) and trichome-specific (GhLTP3) promoters. Transgenic tobacco plants were developed via Agrobacterium-mediated transformation. The phenotypic characteristics of the plants were observed after confirming the integration of transgene in plants. It was observed that CpPIP2 expression cassette under 2×35S and GhLTP3 promoter enhanced the numbers of stem and leave trichomes. However, 2×35S::CpPIP2 has a more amplified effect on trichome density and length than GhLTP3::CpPIP2 and other PIP constructs. These findings imply the role of C. procera PIP aquaporins in fiber cell elongation. The PIPs-derived cell expansion mechanism may be exploited through transgenic approaches for improvement of fiber staple length in cotton and boosting of defense against sucking insects by enhancing plant pubescence.

Molecular characterization and transcriptome profiling of expansin genes isolated from Calotropis procera fibers

The Calotropis procera seed fibers provide an excellent model system to study the genes involved in fiber elongation, fineness and strength. Expansins constitute one of the important gene families involved in plant cell expansion and other cell wall modification processes. Four homologs of Expansin A gene i.e. CpEXPA1, CpEXPA2, CpEXPA3 and CpEXPA4 were isolated from the cDNA library obtained from fast growing Calotropis procera fibers. These homologs represented typical Expansin A family. Each of them had two conserved domains including GH45 like domain and the putative polysaccharide binding domain. The deduced amino acid sequences of the homologs indicated three conserved motifs: i) eight cysteine residues at N-terminus, ii) four tryptophan residues at C-terminus and iii) a Histidine-Phenylalanine-Aspartate motif in the center of the sequence. The presence of N-terminal signal peptide consisting of hydrophobic amino acids and a transmembrane region in all these expansin isoforms suggests their cotranslational insertion into the endoplasmic reticulum and then transportation to the cell wall by secretory pathway. The relative quantification of the four expansins in root, stem, fiber and leave tissues indicated that the transcripts of CpEXPA1, CpEXPA2, CpEXPA3 and CpEXPA4 are variably transcribed in these tissues. The lowest transcription of all the four Expansin A isoforms was observed in elongating roots indicating that root tissue might be having specific expansins other than those confined to air grown organs.

Transient expression of ßC1 protein differentially regulates host genes related to stress response, chloroplast and mitochondrial functions.

BACKGROUND: Geminiviruses are emerging plant pathogens that infect a wide variety of crops including cotton, cassava, vegetables, ornamental plants and cereals. The geminivirus disease complex consists of monopartite begomoviruses that require betasatellites for the expression of disease symptoms. These complexes are widespread throughout the Old World and cause economically important diseases on several crops. A single protein encoded by betasatellites, termed ßC1, is a suppressor of gene silencing, inducer of disease symptoms and is possibly involved in virus movement. Studies of the interaction of ßC1 with hosts can provide useful insight into virus-host interactions and aid in the development of novel control strategies. We have used the differential display technique to isolate host genes which are differentially regulated upon transient expression of the ßC1 protein of chili leaf curl betasatellite (ChLCB) in Nicotiana tabacum. RESULTS: Through differential display analysis, eight genes were isolated from Nicotiana tabacum, at two and four days after infiltration with ßC1 of ChLCB, expressed under the control of the Cauliflower mosaic virus 35S promoter. Cloning and sequence analysis of differentially amplified products suggested that these genes were involved in ATP synthesis, and acted as electron carriers for respiration and photosynthesis processes. These differentially expressed genes (DEGs) play an important role in plant growth and development, cell protection, defence processes, replication mechanisms and detoxification responses. Kegg orthology based annotation system analysis of these DEGs demonstrated that one of the genes, coding for polynucleotide nucleotidyl transferase, is involved in purine and pyrimidine metabolic pathways and is an RNA binding protein which is involved in RNA degradation. CONCLUSION: ßC1 differentially regulated genes are mostly involved in chloroplast and mitochondrial functions. ßC1 also increases the expression of those genes which are involved in purine and pyrimidine metabolism. This information gives a new insight into the interaction of ßC1 with the host and can be used to understand host-virus interactions in follow-up studies.

Identification of differentially expressed genes in developing cotton fibers (Gossypium hirsutum L) through differential display

Cotton fibers are differentiated, non-dividing cells that originate from the epidermal layer of developing ovules. To identify genes involved in cotton fiber development, we performed non-radioactive differential display reverse transcriptase PCR (DDRT-PCR) on the purified mRNA. This technique was tested on mRNA isolated from five different developmental stages of cotton fiber including 0, 5, 10, 15 and 20 DPA (days after pollination). The mRNA purified from total RNA was reversibly transcribed using three anchored oligo-dT primers. Polymerase chain reaction (PCR) amplification of each cDNA preparation was carried out in combination with seven arbitrary primers. The amplified products were resolved on 1 percent agarose gel containing ethidium bromide. DNA was extracted from seventeen differentially expressed bands and cloned in pTZ57R/T vector. The sequencing and BLAST search analysis indicated that 12 of the differentially expressed genes matched the previously characterized genes, while 3 of them matched the uncharacterized sequences of cotton fiber expressed sequence tags (ESTs) reported previously to be associated with cotton fiber and 2 of the clones had homology with putative proteins. The technique can be used to efficiently identify differentially expressed genes and can be expanded to large scale studies by increasing the number of random decamers.