RNAi-mediated down-regulation of ITPK-2 enhanced inorganic phosphorus and minerals in the transgenic rice.

Phytic acid or Myo-inositol hexakisphosphate is an essential compound for the rice plants. It remains in the form of phytate, a mixed salt of different mineral cations, in the seeds. The phytate breaks down during germination and provides the inorganic phosphorus and mineral ions to the seedlings. However, humans do not get the benefit of those essential ions from rice consumption due to the absence of phytase in the gut. We envisaged down-regulating ITPK, the gene behind the phytic acid biosynthesis so that its low amount would facilitate a greater amount of free mineral ions in the endosperm. Since there are six homologues of rice ITPK, we studied their expression in seeds. Additionally, we undertook an in-silico analysis of the homologous proteins. Considering the results, we selected ITPK-2 for its RNAi-mediated embryo-specific down-regulation to obtain the low phytate rice. We obtained a 37% reduction of phytic acid content accompanied by a nearly three-fold enhancement of inorganic phosphorus in the transgenic seeds. Additionally, the iron and zinc content increased in polished rice grains compared to the wild type. The results also showed that reduced phytic acid content did not affect the germination potential and seedling growth of the transgenic rice.

Proteo-metabolomic investigation of transgenic rice unravels metabolic alterations and accumulation of novel proteins potentially involved in defence against Rhizoctonia solani.

The generation of sheath blight (ShB)-resistant transgenic rice plants through the expression of Arabidopsis NPR1 gene is a significant development for research in the field of biotic stress. However, to our knowledge, regulation of the proteomic and metabolic networks in the ShB-resistant transgenic rice plants has not been studied. In the present investigation, the relative proteome and metabolome profiles of the non-transformed wild-type and the AtNPR1-transgenic rice lines prior to and subsequent to the R. solani infection were investigated. Total proteins from wild type and transgenic plants were investigated using two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS). The metabolomics study indicated an increased abundance of various metabolites, which draws parallels with the proteomic analysis. Furthermore, the proteome data was cross-examined using network analysis which identified modules that were rich in known as well as novel immunity-related prognostic proteins, particularly the mitogen-activated protein kinase 6, probable protein phosphatase 2C1, probable trehalose-phosphate phosphatase 2 and heat shock protein. A novel protein, 14-3-3GF14f was observed to be upregulated in the leaves of the transgenic rice plants after ShB infection, and the possible mechanistic role of this protein in ShB resistance may be investigated further.

Development of a rapid and highly efficient Agrobacterium-mediated transformation system for pigeon pea [Cajanus cajan (L.) Millsp].

An efficient genetic transformation system is a prerequisite for studying gene functions, molecular breeding program, and introducing new traits. Agrobacterium tumefaciens-mediated genetic transformation is a widely preferred and accepted method for many plants, including pigeon pea. However, the efficiency of transformation of pigeon pea using the existing protocols is low and time-consuming. In the present study, we developed a rapid and highly efficient transformation system of pigeon pea, using embryonic axis-attached cotyledons as explants. We systematically investigated the influence of varying optical densities of Agrobacterium suspension, duration of incubation, and co-cultivation on the transformation efficiency. In our system, a transformation efficiency of approximately 83% was achieved using Agrobacterium cells at an optical density (OD600) of 0.25, infection time of 15 min, and co-culturing with explants for 72 h in the light with 100µM acetosyringone. The entire procedure, starting from seed to establishment of transformed plants in soil, was achieved in 35-40 days. This is a rapid and highly efficient protocol for Agrobacterium-mediated transformation of pigeon pea, which could potentially be a useful reference, not only for the genetic improvement of pigeon pea but also for other recalcitrant leguminous plants.

Dual gene expression cassette is superior than single gene cassette for enhancing sheath blight tolerance in transgenic rice.

Sheath blight, caused by the necrotrophic fungal pathogen Rhizoctonia solani, is a serious and destructive disease of the rice. In order to improve sheath blight resistance, we developed three different kinds of transgenic rice lines. The first transgenic line overexpresses the rice chitinase gene (OsCHI11); the second contains the Arabidopsis NPR1 (AtNPR1) gene and, the third has pyramided constructs with both the genes (OsCHI11 and AtNPR1). This is a comparative study between the single-gene transgenic lines and the double gene transgenic in terms of their ability to activate the plant defense system. Rice plants of each individual construct were screened via PCR, Southern hybridization, activity assays, and expression analysis. The best transgenic lines of each construct were chosen for comparative study. The fold change in qRT-PCR and activity assays revealed that the pyramided transgenic rice plants show a significant upregulation of defense-related genes, PR genes, and antioxidant marker genes as compared to the single transgene. Simultaneous co-expression of both the genes was found to be more efficient in tolerating oxidative stress. In R. solani (RS) toxin assay, mycelial agar disc bioassay, and in vivo plant bioassay, pyramided transgenic plant lines were more competent at restricting the pathogen development and enhancing sheath blight tolerance as compared to single gene transformants.

Tissue-specific expression of Arabidopsis NPR1 gene in rice for sheath blight resistance without compromising phenotypic cost.

Rice sheath blight disease, caused by the fungus Rhizoctonia solani, is considered the second most important disease of rice after blast. NPR1 (non expressor of PR1) is the central regulator of systemic acquired resistance (SAR) conferring broad spectrum resistance to various pathogens. Previous reports have indicated that constitutive expression of the Arabidopsis thaliana NPR1 (AtNPR1) gene results in disease resistance in rice but has a negative impact on growth and agronomic traits. Here, we report that green tissue-specific expression of AtNPR1 in rice confers resistance to the sheath blight pathogen, with no concomitant abnormalities in plant growth and yield parameters. Elevated levels of NPR1 activated the defence pathway in the transgenic plants by inducing expression of endogenous genes such as PR1b, RC24, and PR10A. Enhanced sheath blight resistance of the transgenic plants was evaluated using three different bioassay systems. A partially isolated toxin from R. solani was used in the bioassays to measure the resistance level. Studies of the phenotype and yield showed that the transgenic plants did not exhibit any kind of phenotypic imbalances. Our results demonstrate that green tissue-specific expression of AtNPR1 is an effective strategy for controlling the sheath blight pathogen. The present work in rice can be extended to other crop plants severely damaged by the pathogen.

Comparative nutritional compositions and proteomics analysis of transgenic Xa21 rice seeds compared to conventional rice.

Transgenic rice expressing the Xa21 gene have enhanced resistant to most devastating bacterial blight diseases caused by Xanthomonas oryzae pv. oryzae (Xoo). However, identification of unintended modifications, owing to the genetic modification, is an important aspect of transgenic crop safety assessment. In this study, the nutritional compositions of seeds from transgenic rice plants expressing the Xa21 gene were compared against non-transgenic rice seeds. In addition, to detect any changes in protein translation levels as a result of Xa21 gene expression, rice seed proteome analyses were also performed by two-dimensional gel electrophoresis. No significant differences were found in the nutritional compositions (proximate components, amino acids, minerals, vitamins and anti-nutrients) of the transgenic and non-transgenic rice seeds. Although gel electrophoresis identified 11 proteins that were differentially expressed between the transgenic and non-transgenic seed, only one of these (with a 20-fold up-regulation in the transgenic seed) shows nutrient reservoir activity. No new toxins or allergens were detected in the transgenic seeds.

Green tissue-specific co-expression of chitinase and oxalate oxidase 4 genes in rice for enhanced resistance against sheath blight.

MAIN CONCLUSION: Green tissue-specific simultaneous overexpression of two defense-related genes ( OsCHI11 & OsOXO4 ) in rice leads to significant resistance against sheath blight pathogen ( R. solani ) without distressing any agronomically important traits. Overexpressing two defense-related genes (OsOXO4 and OsCHI11) cloned from rice is effective at enhancing resistance against sheath blight caused by Rhizoctonia solani. These genes were expressed under the control of two different green tissue-specific promoters, viz. maize phosphoenolpyruvate carboxylase gene promoter, PEPC, and rice cis-acting 544-bp DNA element, immediately upstream of the D54O translational start site, P D54O-544 . Putative T0 transgenic rice plants were screened by PCR and integration of genes was confirmed by Southern hybridization of progeny (T1) rice plants. Successful expression of OsOXO4 and OsCHI11 in all tested plants was confirmed. Expression of PR genes increased significantly following pathogen infection in overexpressing transgenic plants. Following infection, transgenic plants exhibited elevated hydrogen peroxide levels, significant changes in activity of ROS scavenging enzymes and reduced membrane damage when compared to their wild-type counterpart. In a Rhizoctonia solani toxin assay, a detached leaf inoculation test and an in vivo plant bioassay, transgenic plants showed a significant reduction in disease symptoms in comparison to non-transgenic control plants. This is the first report of overexpression of two different PR genes driven by two green tissue-specific promoters providing enhanced sheath blight resistance in transgenic rice.

Down-regulation of lipoxygenase gene reduces degradation of carotenoids of golden rice during storage.

MAIN CONCLUSION: Down-regulation of lipoxygenase enzyme activity reduces degradation of carotenoids of bio-fortified rice seeds which would be an effective tool to reduce huge post-harvest and economic losses of bio-fortified rice seeds during storage. Bio-fortified provitamin A-enriched rice line (golden rice) expressing higher amounts of ß-carotene in the rice endosperm provides vitamin A for human health. However, it is already reported that degradation of carotenoids during storage is a major problem. The gene responsible for degradation of carotenoids during storage has remained largely unexplored till now. In our previous study, it has been shown that r9-LOX1 gene is responsible for rice seed quality deterioration. In the present study, we attempted to investigate if r9-LOX1 gene has any role in degradation of carotenoids in rice seeds during storage. To establish our hypothesis, the endogenous lipoxygenase (LOX) activity of high-carotenoid golden indica rice seed was silenced by RNAi technology using aleurone layer and embryo-specific Oleosin-18 promoter. To check the storage stability, LOX enzyme down-regulated high-carotenoid T3 transgenic rice seeds were subjected to artificial aging treatment. The results obtained from biochemical assays (MDA, ROS) also indicated that after artificial aging, the deterioration of LOX-RNAi lines was considerably lower compared to ß-carotene-enriched transgenic rice which had higher LOX activity in comparison to LOX-RNAi lines. Furthermore, it was also observed by HPLC analysis that down-regulation of LOX gene activity decreases co-oxidation of ß-carotene in LOX-RNAi golden rice seeds as compared to the ß-carotene-enriched transgenic rice, after artificial aging treatment. Therefore, our study substantially establishes and verifies that LOX is a key enzyme for catalyzing co-oxidation of ß-carotene and has a significant role in deterioration of ß-carotene levels in the carotenoid-enriched golden rice.

RNAi mediated down regulation of myo-inositol-3-phosphate synthase to generate low phytate rice.

BACKGROUND: Phytic acid (InsP6) is considered as the major source of phosphorus and inositol phosphates in cereal grains. Reduction of phytic acid level in cereal grains is desirable in view of its antinutrient properties to maximize mineral bioavailability and minimize the load of phosphorus waste management. We report here RNAi mediated seed-specific silencing of myo-inositol-3-phosphate synthase (MIPS) gene catalyzing the first step of phytic acid biosynthesis in rice. Moreover, we also studied the possible implications of MIPS silencing on myo-inositol and related metabolism, since, first step of phytic acid biosynthesis is also the rate limiting step of myo-inositol synthesis, catalyzed by MIPS. RESULTS: The resulting transgenic rice plants (T3) showed a 4.59 fold down regulation in MIPS gene expression, which corresponds to a significant decrease in phytate levels and a simultaneous increment in the amount of inorganic phosphate in the seeds. A diminution in the myo-inositol content of transgenic plants was also observed due to disruption of the first step of phytic acid biosynthetic pathway, which further reduced the level of ascorbate and altered abscisic acid (ABA) sensitivity of the transgenic plants. In addition, our results shows that in the transgenic plants, the lower phytate levels has led to an increment of divalent cations, of which a 1.6 fold increase in the iron concentration in milled rice seeds was noteworthy. This increase could be attributed to reduced chelation of divalent metal (iron) cations, which may correlate to higher iron bioavailability in the endosperm of rice grains. CONCLUSION: The present study evidently suggests that seed-specific silencing of MIPS in transgenic rice plants can yield substantial reduction in levels of phytic acid along with an increase in inorganic phosphate content. However, it was also demonstrated that the low phytate seeds had an undesirable diminution in levels of myo-inositol and ascorbate, which probably led to sensitiveness of seeds to abscisic acid during germination. Therefore, it is suggested that though MIPS is the prime target for generation of low phytate transgenic plants, down-regulation of MIPS can have detrimental effect on myo-inositol synthesis and related pathways which are involved in key plant metabolism.

Development of low phytate rice by RNAi mediated seed-specific silencing of inositol 1,3,4,5,6-pentakisphosphate 2-kinase gene (IPK1).

Phytic acid (InsP(6)) is considered to be the major source of phosphorus and inositol phosphates in most cereal grains. However, InsP(6) is not utilized efficiently by monogastric animals due to lack of phytase enzyme. Furthermore, due to its ability to chelate mineral cations, phytic acid is considered to be an antinutrient that renders these minerals unavailable for absorption. In view of these facts, reducing the phytic acid content in cereal grains is a desired goal for the genetic improvement of several crops. In the present study, we report the RNAi-mediated seed-specific silencing (using the Oleosin18 promoter) of the IPK1 gene, which catalyzes the last step of phytic acid biosynthesis in rice. The presence of the transgene cassette in the resulting transgenic plants was confirmed by molecular analysis, indicating the stable integration of the transgene. The subsequent T4 transgenic seeds revealed 3.85-fold down-regulation in IPK1 transcripts, which correlated to a significant reduction in phytate levels and a concomitant increase in the amount of inorganic phosphate (Pi). The low-phytate rice seeds also accumulated 1.8-fold more iron in the endosperm due to the decreased phytic acid levels. No negative effects were observed on seed germination or in any of the agronomic traits examined. The results provide evidence that silencing of IPK1 gene can mediate a substantial reduction in seed phytate levels without hampering the growth and development of transgenic rice plants.

Comparative analysis of nutritional compositions of transgenic high iron rice with its non-transgenic counterpart.

Iron is an essential micronutrient for human nutrition and polished rice contains very low amount of iron. Rice with high iron content in seed endosperm has been developed by insertion of soybean ferritin gene under the control of the endosperm specific glutelin promoter into the genome of indica rice line IR68144. The nutritional composition of the brown and milled rice grain has been compared with that of the non-transgenic rice of the same variety. In this study, the nutritional components, as well as the anti-nutrient levels, were measured. Our studies established that apart from the increased level of iron and zinc in transgenic seeds, the nutritional quality of both the brown and milled rice grains from the transgenic line was substantially equivalent to that of the non-transgenic rice plants. The result clearly shows that the measured amounts of the nutritional components are well within the range of values reported for other commercial lines.

Sporulation in mycobacteria.

Mycobacteria owe their success as pathogens to their ability to persist for long periods within host cells in asymptomatic, latent forms before they opportunistically switch to the virulent state. The molecular mechanisms underlying the transition into dormancy and emergence from it are not clear. Here we show that old cultures of Mycobacterium marinum contained spores that, upon exposure to fresh medium, germinated into vegetative cells and reappeared again in stationary phase via endospore formation. They showed many of the usual characteristics of well-known endospores. Homologues of well-known sporulation genes of Bacillus subtilis and Streptomyces coelicolor were detected in mycobacteria genomes, some of which were verified to be transcribed during appropriate life-cycle stages. We also provide data indicating that it is likely that old Mycobacterium bovis bacillus Calmette-Guérin cultures form spores. Together, our data show sporulation as a lifestyle adapted by mycobacteria under stress and tempt us to suggest this as a possible mechanism for dormancy and/or persistent infection. If so, this might lead to new prophylactic strategies.

A dideoxynucleotide-sensitive DNA polymerase activity characterized from endoreduplicating cells of mungbean (Vigna radiata L.) during ontogeny of cotyledons.

Within this work we describe the purification and biochemical characterization of a ddNTP-sensitive DNA polymerase purified from mungbean (Vigna radiata cv B1, L.) seeds at 18 days after fertilization, when > 70% of the nuclei are reported to be in the endoreduplicated state. The purified enzyme is a single polypeptide of 62 kDa and many of its physicochemical properties are similar to those of mammalian DNA polymerase beta. Similar to the other X-family DNA polymerases, it lacks 3'-5' exonuclease activity and has short gap-filling and strand-displacement activity. The enzyme shows moderately processive DNA synthesis on a single-strand template. The determined N-terminal heptapeptide sequence of the enzyme showed clear homology with helix 1 of the N-terminal single strand DNA-binding domain (residues 32-41) of rat and human DNA polymerase beta. These results represent the first evidence for the identification and characterization of a ddNTP-sensitive DNA polymerase expressed during the endoreduplication cycle that shares biochemical and immunological similarity with mammalian DNA polymerase beta.

Dideoxynucleoside triphosphate-sensitive DNA polymerase from rice is involved in base excision repair and immunologically similar to mammalian DNA pol beta.

A single polypeptide with ddNTP-sensitive DNA polymerase activity was purified to near homogeneity from the shoot tips of rice seedlings and analysis of the preparations by SDS-PAGE followed by silver staining showed a polypeptide of 67 kDa size. The DNA polymerase activity was found to be inhibitory by ddNTP in both in vitro DNA polymerase activity assay and activity gel analysis. Aphidicolin, an inhibitor of other types of DNA polymerases, had no effect on plant enzyme. The 67 kDa rice DNA polymerase was found to be recognized by the polyclonal antibody (purified IgG) made against rat DNA polymerase beta (pol beta) both in solution and also on Western blot. The recognition was found to be very specific as the activity of Klenow enzyme was unaffected by the antibody. The ability of rice nuclear extract to correct G:U mismatch of oligo-duplex was observed when oligo-duplex with 32P-labeled lower strand containing U (at 22nd position) was used as substrate. Differential appearance of bands at 21-mer, 22-mer, and 51-mer position in presence of dCTP was visible only with G:U mismatch oligo-duplex, but not with G:C oligo-duplex. While ddCTP or polyclonal antibody against rat-DNA pol beta inhibits base excision repair (BER), aphidicolin had no effect. These results for the first time clearly demonstrate the ability of rice nuclear extract to run BER and the involvement of ddNTP-sensitive pol beta type DNA polymerase. Immunological similarity of the ddNTP-sensitive DNA polymerase beta of rice and rat and its involvement in BER revealed the conservation of structure and function of ddNTP-sensitive DNA pol beta in plant and animal.