Genetic engineering low-arsenic and low-cadmium rice grain.

Rice is prone to take up the toxic elements arsenic (As) and cadmium (Cd) from paddy soil through the transporters for other essential elements. Disruption of these essential transporters usually adversely affects the normal growth of rice and the homeostasis of essential elements. Here we report on developing low-As and low-Cd rice grain through the co-overexpression of OsPCS1, OsABCC1, and OsHMA3 genes under the control of the rice OsActin1 promoter. Co-overexpression of OsPCS1 and OsABCC1 synergistically decreased As concentration in the grain. Overexpression of OsPCS1 also decreased Cd concentration in the grain by restricting the xylem-to-phloem Cd transport in node I, but paradoxically caused Cd hypersensitivity as the overproduced phytochelatins in OsPCS1-overexpressing plants suppressed OsHMA3-dependent Cd sequestration in vacuoles and promoted Cd transport from root to shoot. Co-overexpression of OsHAM3 and OsPCS1 overcame this suppression and complemented the Cd hypersensitivity. Compared with non-transgenic rice control, co-overexpression of OsABCC1, OsPCS1, and OsHMA3 in rice decreased As and Cd concentrations in grain by 92.1% and 98%, respectively, without causing any defect in plant growth and reproduction or of mineral nutrients in grain. Our research provides an effective approach and useful genetic materials for developing low-As and low-Cd rice grain.

TAL effector-dependent Bax gene expression in transgenic rice confers disease resistance to Xanthomonas oryzae pv. oryzae.

The hypersensitive response (HR) is a form of programmed cell death of plant cells occurring in the local region surrounding pathogen infection site to prevent the spread of infection by pathogens. Bax, a mammalian pro-apoptotic member of Bcl-2 family, triggers HR-like cell death when expressed in plants. However, constitutive expression of the Bax gene negatively affects plant growth and development. The Xa10 gene in rice (Oryza sativa) is an executor resistance (R) gene that confers race-specific disease resistance to Xanthomonas oryzae pv. oryzae strains harboring TAL effector gene AvrXa10. In this study, the Xa10 promoter was used to regulate heterologous expression of the Bax gene from mouse (Mus musculus) in Nicotiana benthamiana and rice. Cell death was induced in N. benthamiana after co-infiltration with the PXa10:Bax:TXa10 gene and the PPR1:AvrXa10:TNos gene. Transgenic rice plants carrying the PXa10:Bax:TXa10 gene conferred specific disease resistance to Xa10-incompatible X. oryzae pv. oryzae strain PXO99A(pHM1AvrXa10), but not to the Xa10-compatible strain PXO99A(pHM1). The resistance specificity was confirmed by the AvrXa10-dependent induction of the PXa10:Bax:TXa10 gene in transgenic rice. Our results demonstrated that the inducible expression of the Bax gene in transgenic rice was achieved through the control of the executor R gene promoter and the heterologous expression of the pro-apoptosis regulator gene in rice conferred disease resistance to X. oryzae pv. oryzae.

The pepper Bs4C proteins are localized to the endoplasmic reticulum (ER) membrane and confer disease resistance to bacterial blight in transgenic rice.

Transcription activator-like effector (TALE)-dependent dominant disease resistance (R) genes in plants, also referred to as executor R genes, are induced on infection by phytopathogenic bacteria of the genus Xanthomonas harbouring the corresponding TALE genes. Unlike the traditional R proteins, the executor R proteins do not determine the resistance specificity and may function broadly in different plant species. The executor R gene Bs4C-R in the resistant genotype PI 235047 of the pepper species Capsicum pubescens (CpBs4C-R) confers disease resistance to Xanthomonas campestris pv. vesicatoria (Xcv) harbouring the TALE genes avrBsP/avrBs4. In this study, the synthetic genes of CpBs4C-R and two other Bs4C-like genes, the susceptible allele in the genotype PI585270 of C. pubescens (CpBs4C-S) and the CaBs4C-R homologue gene in the cultivar 'CM334' of Capsicum annum (CaBs4C), were characterized in tobacco (Nicotiana benthamiana) and rice (Oryza sativa). The Bs4C genes induced cell death in N. benthamiana. The functional Bs4C-eCFP fusion proteins were localized to the endoplasmic reticulum (ER) membrane in the leaf epidermal cells of N. benthamiana. The Xa10 promoter-Bs4C fusion genes in transgenic rice conferred strain-specific disease resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight in rice, and were specifically induced by the Xa10-incompatible Xoo strain PXO99A (pHM1avrXa10). The results indicate that the Bs4C proteins from pepper species function broadly in rice and the Bs4C protein-mediated cell death from the ER is conserved between dicotyledonous and monocotyledonous plants, which can be utilized to engineer novel and enhanced disease resistance in heterologous plants.

Induction of Xa10-like Genes in Rice Cultivar Nipponbare Confers Disease Resistance to Rice Bacterial Blight.

Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae, is one of the most destructive bacterial diseases throughout the major rice-growing regions in the world. The rice disease resistance (R) gene Xa10 confers race-specific disease resistance to X. oryzae pv. oryzae strains that deliver the corresponding transcription activator-like (TAL) effector AvrXa10. Upon bacterial infection, AvrXa10 binds specifically to the effector binding element in the promoter of the R gene and activates its expression. Xa10 encodes an executor R protein that triggers hypersensitive response and activates disease resistance. 'Nipponbare' rice carries two Xa10-like genes in its genome, of which one is the susceptible allele of the Xa23 gene, a Xa10-like TAL effector-dependent executor R gene isolated recently from 'CBB23' rice. However, the function of the two Xa10-like genes in disease resistance to X. oryzae pv. oryzae strains has not been investigated. Here, we designated the two Xa10-like genes as Xa10-Ni and Xa23-Ni and characterized their function for disease resistance to rice bacterial blight. Both Xa10-Ni and Xa23-Ni provided disease resistance to X. oryzae pv. oryzae strains that deliver the matching artificially designed TAL effectors (dTALE). Transgenic rice plants containing Xa10-Ni and Xa23-Ni under the Xa10 promoter provided specific disease resistance to X. oryzae pv. oryzae strains that deliver AvrXa10. Xa10-Ni and Xa23-Ni knock-out mutants abolished dTALE-dependent disease resistance to X. oryzae pv. oryzae. Heterologous expression of Xa10-Ni and Xa23-Ni in Nicotiana benthamiana triggered cell death. The 19-amino-acid residues at the N-terminal regions of XA10 or XA10-Ni are dispensable for their function in inducing cell death in N. benthamiana and the C-terminal regions of XA10, XA10-Ni, and XA23-Ni are interchangeable among each other without affecting their function. Like XA10, both XA10-Ni and XA23-Ni locate to the endoplasmic reticulum (ER) membrane, show self-interaction, and induce ER Ca2+ depletion in leaf cells of N. benthamiana. The results indicate that Xa10-Ni and Xa23-Ni in Nipponbare encode functional executor R proteins, which induce cell death in both monocotyledonous and dicotyledonous plants and have the potential of being engineered to provide broad-spectrum disease resistance to plant-pathogenic Xanthomonas spp.

Engineering low phorbol ester Jatropha curcas seed by intercepting casbene biosynthesis.

KEY MESSAGE: Casbene is a precursor to phorbol esters and down-regulating casbene synthase effectively reduces phorbol ester biosynthesis. Seed-specific reduction of phorbol ester (PE) helps develop Jatropha seed cake for animal nutrition. Phorbol esters (PEs) are diterpenoids present in some Euphorbiaceae family members like Jatropha curcas L. (Jatropha), a tropical shrub yielding high-quality oil suitable as feedstock for biodiesel and bio jet fuel. Jatropha seed contains up to 40 % of oil and can produce oil together with cake containing high-quality proteins. However, skin-irritating and cancer-promoting PEs make Jatropha cake meal unsuitable for animal nutrition and also raise some safety and environmental concerns on its planting and processing. Two casbene synthase gene (JcCASA163 and JcCASD168) homologues were cloned from Jatropha genome and both genes were highly expressed during seed development. In vitro functional analysis proved casbene synthase activity of JcCASA163 in converting geranylgeranyl diphosphate into casbene which has been speculated to be the precursor to PEs. A seed-specific promoter driving inverted repeats for RNAi interference targeting at either JcCASA163 or both genes could effectively down-regulate casbene synthase gene expression with concurrent marked reduction of PE level (by as much as 85 %) in seeds with no pleiotropic effects observed. Such engineered low PE in seed was heritable and co-segregated with the transgene. Our work implicated casbene synthase in Jatropha PE biosynthesis and provided evidence for casbene being the precursor for PEs. The success in reducing seed PE content through down-regulation of casbene synthase demonstrates the feasibility of intercepting PE biosynthesis in Jatropha seed to help address safety concerns on Jatropha plantation and seed processing and facilitate use of its seed protein for animal nutrition.

The rice TAL effector-dependent resistance protein XA10 triggers cell death and calcium depletion in the endoplasmic reticulum.

The recognition between disease resistance (R) genes in plants and their cognate avirulence (Avr) genes in pathogens can produce a hypersensitive response of localized programmed cell death. However, our knowledge of the early signaling events of the R gene-mediated hypersensitive response in plants remains limited. Here, we report the cloning and characterization of Xa10, a transcription activator-like (TAL) effector-dependent R gene for resistance to bacterial blight in rice (Oryza sativa). Xa10 contains a binding element for the TAL effector AvrXa10 (EBEAvrXa10) in its promoter, and AvrXa10 specifically induces Xa10 expression. Expression of Xa10 induces programmed cell death in rice, Nicotiana benthamiana, and mammalian HeLa cells. The Xa10 gene product XA10 localizes as hexamers in the endoplasmic reticulum (ER) and is associated with ER Ca(2+) depletion in plant and HeLa cells. XA10 variants that abolish programmed cell death and ER Ca(2+) depletion in N. benthamiana and HeLa cells also abolish disease resistance in rice. We propose that XA10 is an inducible, intrinsic terminator protein that triggers programmed cell death by a conserved mechanism involving disruption of the ER and cellular Ca(2+) homeostasis.

An improved method for RNA isolation and cDNA library construction from immature seeds of Jatropha curcas L.

BACKGROUND: RNA quality and quantity is sometimes unsuitable for cDNA library construction, from plant seeds rich in oil, polysaccharides and other secondary metabolites. Seeds of jatropha (Jatropha curcas L.) are rich in fatty acids/lipids, storage proteins, polysaccharides, and a number of other secondary metabolites that could either bind and/or co-precipitate with RNA, making it unsuitable for downstream applications. Existing RNA isolation methods and commercial kits often fail to deliver high-quality total RNA from immature jatropha seeds for poly(A)+ RNA purification and cDNA synthesis. FINDINGS: A protocol has been developed for isolating good quality total RNA from immature jatropha seeds, whereby a combination of the CTAB based RNA extraction method and a silica column of a commercial plant RNA extraction kit is used. The extraction time was reduced from two days to about 3 hours and the RNA was suitable for poly(A)+ RNA purification, cDNA synthesis, cDNA library construction, RT-PCR, and Northern hybridization. Based on sequence information from selected clones and amplified PCR product, the cDNA library seems to be a good source of full-length jatropha genes. The method was equally effective for isolating RNA from mustard and rice seeds. CONCLUSIONS: This is a simple CTAB + silica column method to extract high quality RNA from oil rich immature jatropha seeds that is suitable for several downstream applications. This method takes less time for RNA extraction and is equally effective for other tissues where the quality and quantity of RNA is highly interfered by the presence of fatty acids, polysaccharides and polyphenols.