Performance evaluation and comparative research of underwater wireless optical communication system by using different structured beams.

Structured beams have attracted increasing interest in free-space and fiber-based optical communications. Underwater wireless optical communication (UWOC) is becoming a prospective technique in marine exploration. We investigated UWOC performance using different representative structured beams. The transmission performances of the Gaussian, Bessel-Gaussian (BG), Ince-Gaussian (IG), and radially polarized Gaussian (RPG) beams were experimentally demonstrated and evaluated in underwater channels subjected to thermal gradient. The experimental results show that the BG, IG, and RPG perform better against the thermal gradient. Compared with the Gaussian beams, the beam wanders of BG, IG, and RPG beams under the thermal gradient have been reduced by 56.9%, 8.2%, and 59%, the scintillation indices have been decreased by 12.8%, 17.3%, and 28.9%, and the BER performance of the BG, IG, and RPG beams have been improved by ∼5.5, ∼3.7, and ∼5.2d B at the forward error correction threshold (FEC threshold). Based on the above results, the RPG beam is a more promising light source for UWOC. The experimental results provide a promising beam choice for UWOC.

Ectopic Expression of the Executor-Type R Gene Paralog Xa27B in Rice Leads to Spontaneous Lesions and Enhanced Disease Resistance.

Plant disease resistance (R) gene-mediated effector-triggered immunity (ETI) is usually associated with hypersensitive response (HR) and provides robust and race-specific disease resistance against pathogenic infection. The activation of ETI and HR in plants is strictly regulated, and improper activation will lead to cell death. Xa27 is an executor-type R gene in rice induced by the TAL effector AvrXa27 and confers disease resistance to Xanthomonas oryzae pv. oryzae (Xoo). Here we reported the characterization of a transgenic line with lesion mimic phenotype, designated as Spotted leaf and resistance 1 (Slr1), which was derived from rice transformation with a genomic subclone located 5,125 bp downstream of the Xa27 gene. Slr1 develops spontaneous lesions on its leaves caused by cell death and confers disease resistance to both Xoo and Xanthomonas oryzae pv. oryzicola. Further investigation revealed that the Slr1 phenotype resulted from the ectopic expression of an Xa27 paralog gene, designated as Xa27B, in the inserted DNA fragment at the Slr1 locus driven by a truncated CaMV35Sx2 promoter in reverse orientation. Disease evaluation of IRBB27, IR24, and Xa27B mutants with Xoo strains expressing dTALE-Xa27B confirmed that Xa27B is a functional executor-type R gene. The functional XA27B-GFP protein was localized to the endoplasmic reticulum and apoplast. The identification of Xa27B as a new functional executor-type R gene provides additional genetic resources for studying the mechanism of executor-type R protein-mediated ETI and developing enhanced and broad-spectrum disease resistance to Xoo through promoter engineering. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

Inactivation of retrotransposon Tos17 Chr.7 in rice cultivar Nipponbare through CRISPR/Cas9-mediated gene editing.

Retrotransposons are mobile genetic elements capable of transposition via reverse transcription of RNA intermediates. Rice cultivar Nipponbare contains two nearly identical genomic copies of Tos17, an endogenous copia-like LTR retrotransposon, on chromosomes 7 (Tos17 Chr.7) and 10 (Tos17 Chr.10), respectively. Previous studies demonstrated that only Tos17 Chr.7 is active in transposition during tissue culture. Tos17 Chr.7 has been extensively used for insertional mutagenesis as a tool for functional analysis of rice genes. However, Tos17 Chr.7 transposition might generate somaclonal mutagenesis with undesirable traits during rice transformation, which would affect the evaluation or application of transgenes. In this study, we generated a Tos17 Chr.7 knockout mutant D873 by using CRISPR/Cas9 gene editing system. The gene-edited allele of Tos17 Chr.7 in D873, designated as Tos17 D873, has an 873-bp DNA deletion in the pol gene of Tos17 Chr.7, which caused the deletion of the GAG-pre-integrase domain and the integrase core domain. Although the transcription of Tos17 D873 was activated in D873 calli, no transposition of Tos17 D873 was detected in the regenerated D873 plants. The results demonstrate that the GAG-pre-integrase domain and the integrase core domain are essential for Tos17 Chr.7 transposition and the deletion of the two domains could be not complemented by other LTR retrotransposons in rice genome. As the Tos17 Chr.7-derived somaclonal mutagenesis is blocked in the D873 plants, the generation of the Tos17 D873 allele will be helpful in production of transgenic rice plants for gene function study and genetic engineering. Similar approach can be used to inactivate other retrotransposons in crop breeding.

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.

Development of marker-free transgenic Jatropha curcas producing curcin-deficient seeds through endosperm-specific RNAi-mediated gene silencing.

BACKGROUND: Jatropha curcas L. is a potential biofuel plant and its seed oil is suitable for biodiesel production. Despite this promising application, jatropha seeds contain two major toxic components, namely phorbol esters and curcins. These compounds would reduce commercial value of seed cake and raise safety and environment concerns on jatropha plantation and processing. Curcins are Type I ribosome inactivating proteins. Several curcin genes have been identified in the jatropha genome. Among which, the Curcin 1 (C1) gene is identified to be specifically expressed in endosperm, whereas the Curcin 2A (C2A) is mainly expressed in young leaves. RESULTS: A marker-free RNAi construct carrying a ß-estradiol-regulated Cre/loxP system and a C1 promoter-driven RNAi cassette for C1 gene was made and used to generate marker-free transgenic RNAi plants to specifically silence the C1 gene in the endosperm of J. curcas. Plants of transgenic line L1, derived from T0-1, carry two copies of marker-free RNAi cassette, whereas plants of L35, derived from T0-35, harbored one copy of marker-free RNAi cassette and three copies of closely linked and yet truncated Hpt genes. The C1 protein content in endosperm of L1 and L35 seeds was greatly reduced or undetectable, while the C2A proteins in young leaves of T0-1 and T0-35 plants were unaffected. In addition, the C1 mRNA transcripts were undetectable in the endosperm of T3 seeds of L1 and L35. The results demonstrated that the expression of the C1 gene was specifically down-regulated or silenced by the double-stranded RNA-mediated RNA interference generated from the RNAi cassette. CONCLUSION: The C1 promoter-driven RNAi cassette for the C1 gene in transgenic plants was functional and heritable. Both C1 transcripts and C1 proteins were greatly down-regulated or silenced in the endosperm of transgenic J. curcas. The marker-free transgenic plants and curcin-deficient seeds developed in this study provided a solution for the toxicity of curcins in jatropha seeds and addressed the safety concerns of the marker genes in transgenic plants on the environments.

Genetic engineering of the Xa10 promoter for broad-spectrum and durable resistance to Xanthomonas oryzae pv. oryzae.

Many pathovars of plant pathogenic bacteria Xanthomonas species inject transcription activator-like (TAL) effectors into plant host cells to promote disease susceptibility or trigger disease resistance. The rice TAL effector-dependent disease resistance gene Xa10 confers narrow-spectrum race-specific resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight disease in rice. To generate broad-spectrum and durable resistance to Xoo, we developed a modified Xa10 gene, designated as Xa10(E5) . Xa10(E5) has an EBE-amended promoter containing 5 tandemly arranged EBEs each responding specifically to a corresponding virulent or avirulent TAL effector and a stable transgenic rice line containing Xa10(E5) was generated in the cultivar Nipponbare. The Xa10(E5) gene was specifically induced by Xoo strains that harbour the corresponding TAL effectors and conferred TAL effector-dependent resistance to the pathogens at all developmental stages of rice. Further disease evaluation demonstrated that the Xa10(E5) gene in either Nipponbare or 9311 genetic backgrounds provided broad-spectrum disease resistance to 27 of the 28 Xoo strains collected from 11 countries. The development of Xa10(E5) and transgenic rice lines provides new genetic materials for molecular breeding of rice for broad-spectrum and durable disease resistance to bacterial blight.

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.

Expression of fatty acid and lipid biosynthetic genes in developing endosperm of Jatropha curcas.

BACKGROUND: Temporal and spatial expression of fatty acid and lipid biosynthetic genes are associated with the accumulation of storage lipids in the seeds of oil plants. In jatropha (Jatropha curcas L.), a potential biofuel plant, the storage lipids are mainly synthesized and accumulated in the endosperm of seeds. Although the fatty acid and lipid biosynthetic genes in jatropha have been identified, the expression of these genes at different developing stages of endosperm has not been systemically investigated. RESULTS: Transmission electron microscopy study revealed that the oil body formation in developing endosperm of jatropha seeds initially appeared at 28 days after fertilization (DAF), was actively developed at 42 DAF and reached to the maximum number and size at 56 DAF. Sixty-eight genes that encode enzymes, proteins or their subunits involved in fatty acid and lipid biosynthesis were identified from a normalized cDNA library of jatropha developing endosperm. Gene expression with quantitative reverse-transcription polymerase chain reaction analysis demonstrated that the 68 genes could be collectively grouped into five categories based on the patterns of relative expression of the genes during endosperm development. Category I has 47 genes and they displayed a bell-shaped expression pattern with the peak expression at 28 or 42 DAF, but low expression at 14 and 56 DAF. Category II contains 8 genes and expression of the 8 genes was constantly increased from 14 to 56 DAF. Category III comprises of 2 genes and both genes were constitutively expressed throughout endosperm development. Category IV has 9 genes and they showed a high expression at 14 and 28 DAF, but a decreased expression from 42 to 56 DAF. Category V consists of 2 genes and both genes showed a medium expression at 14 DAF, the lowest expression at 28 or 42 DAF, and the highest expression at 56 DAF. In addition, genes encoding enzymes or proteins with similar function were differentially expressed during endosperm development. CONCLUSION: The formation of oil bodies in jatropha endosperm is developmentally regulated. The expression of the majority of fatty acid and lipid biosynthetic genes is highly consistent with the development of oil bodies and endosperm in jatropha seeds, while the genes encoding enzymes with similar function may be differentially expressed during endosperm development. These results not only provide the initial information on spatial and temporal expression of fatty acid and lipid biosynthetic genes in jatropha developing endosperm, but are also valuable to identify the rate-limiting genes for storage lipid biosynthesis and accumulation during seed development.

Molecular cloning and expression of heteromeric ACCase subunit genes from Jatropha curcas.

Acetyl-CoA carboxylase (ACCase) catalyzes the biotin-dependent carboxylation of acetyl-CoA to produce malonyl-CoA, which is the essential first step in the biosynthesis of long-chain fatty acids. ACCase exists as a multi-subunit enzyme in most prokaryotes and the chloroplasts of most plants and algae, while it is present as a multi-domain enzyme in the endoplasmic reticulum of most eukaryotes. The heteromeric ACCase of higher plants consists of four subunits: an α-subunit of carboxyltransferase (α-CT, encoded by accA gene), a biotin carboxyl carrier protein (BCCP, encoded by accB gene), a biotin carboxylase (BC, encoded by accC gene) and a ß-subunit of carboxyltransferase (ß-CT, encoded by accD gene). In this study, we cloned and characterized the genes accA, accB1, accC and accD that encode the subunits of heteromeric ACCase in Jatropha (Jatropha curcas), a potential biofuel plant. The full-length cDNAs of the four subunit genes were isolated from a Jatropha cDNA library and by using 5' RACE, whereas the genomic clones were obtained from a Jatropha BAC library. They encode a 771 amino acid (aa) α-CT, a 286-aa BCCP1, a 537-aa BC and a 494-aa ß-CT, respectively. The single-copy accA, accB1 and accC genes are nuclear genes, while the accD gene is located in chloroplast genome. Jatropha α-CT, BCCP1, BC and ß-CT show high identity to their homologues in other higher plants at amino acid level and contain all conserved domains for ACCase activity. The accA, accB1, accC and accD genes are temporally and spatially expressed in the leaves and endosperm of Jatropha plants, which are regulated by plant development and environmental factors.

Production of marker-free transgenic rice expressing tissue-specific Bt gene.

The hybrid Bacillus thuringiensis (Bt) δ-endotoxin gene Cry1Ab/Ac was used to develop a transgenic Bt rice (Oryza sativa L.) targeting lepidopteran insects of rice. Here, we show the production of a marker-free and tissue-specific expressing transgenic Bt rice line L24 using Agrobacterium-mediated transformation and a chemically regulated, Cre/loxP-mediated DNA recombination system. L24 carries a single copy of marker-free T-DNA that contains the Cry1Ab/Ac gene driven by a maize phosphoenolpyruvate carboxylase (PEPC) gene promoter. The marker-free T-DNA was integrated into the 3' untranslated region of rice gene Os01g0154500 on the short arm of chromosome 1. Compared to the constitutive and non-specific expression of the P (Actin1):Cry1Ab/Ac:T (Nos) gene in the control Bt rice line T51-1, the P ( Pepc ):Cry1Ab/Ac:T (Nos ) gene was detected only in the leaf and stem tissues of L24. More importantly, compared to high levels of CRY1Ab/Ac proteins accumulated in T51-1 seeds, the CRY1Ab/Ac proteins were not detectable in L24 seeds by Western blot analysis. As demonstrated by insect bioassay, L24 provided similar level of resistance to rice leaffolder (Cnaphalocrocis medinalis) as T51-1. The marker-free transgenic line L24 can be used directly in rice breeding for insect resistance to lepidopteran insects where absence of Bt toxin protein in the seed is highly desirable.

Transcription activator-like type III effector AvrXa27 depends on OsTFIIAgamma5 for the activation of Xa27 transcription in rice that triggers disease resistance to Xanthomonas oryzae pv. oryzae.

The transcription activator-like (TAL) type III effector AvrXa27 from Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99(A) activates the transcription of the host resistance gene Xa27, which results in disease resistance to bacterial blight (BB) in rice. In this study, we show that AvrXa27-activated Xa27 transcription requires host general transcription factor OsTFIIAgamma5. The V39E substitution in OsTFIIAgamma5, encoded by the recessive resistance gene xa5 in rice, greatly attenuates this activation in xa5 and Xa27 double homozygotes on inoculation with Xa27-incompatible strains. The xa5 gene also causes attenuation in the induction of Xa27 by AvrXa27 expressed in rice. The xa5-mediated attenuation of Xa27-mediated resistance to PXO99(A) is recessive. Intriguingly, xa5-mediated resistance to xa5-incompatible strains is also down-regulated in the xa5 and Xa27 double homozygotes. In addition, AvrXa27 expressed in planta shows weak virulence activity in the xa5 genetic background and causes enhanced susceptibility of the plants to BB inoculation. The results suggest that TAL effectors target host general transcription factors to directly manipulate the host transcriptional machinery for virulence and/or avirulence. The identification of xa5-mediated attenuation of Xa27-mediated resistance to Xoo provides a guideline for breeding resistance to BB when pyramiding xa5 with other resistance genes.

Constitutive heterologous expression of avrXa27 in rice containing the R gene Xa27 confers enhanced resistance to compatible Xanthomonas oryzae strains.

The vascular pathogen Xanthomonas oryzae pv. oryzae (Xoo) and nonvascular pathogen Xanthomonas oryzae pv. oryzicola (Xoc) cause bacterial blight (BB) and bacterial leaf streak (BLS) diseases of rice, respectively. We have previously identified the avirulence gene avrXa27 from Xoo PXO99(A), which specifically induces the expression of the rice resistance gene Xa27, ultimately leading to resistance against BB disease in rice. In this study, we have generated a transgenic rice line (L24) that expresses avrXa27 constitutively under the control of the PR1 promoter, and have examined its role in the host-pathogen interaction. L24 is not more susceptible to BB, indicating that avrXa27 does not contribute to virulence. AvrXa27 retains avirulence activity in L24 and, after crossing with a line containing Xa27, progeny display phenotypic changes including inhibition of tillering, delay in flowering, stiff leaves, early leaf senescence and activation of pathogenesis-related (PR) genes. On challenge with a variety of compatible strains of Xoo and Xoc strain L8, lines with both avrXa27 and Xa27 also show enhanced resistance to bacterial infection. The induction of Xa27 and subsequent inhibition of Xoc growth in Xa27 plants are observed on inoculation with Xoc L8 harbouring avrXa27. Our results indicate that the heterologous expression of avrXa27 in rice containing Xa27 triggers R gene-specific resistance and, at the same time, confers enhanced resistance to compatible strains of Xoo and Xoc. The expression of AvrXa27 and related proteins in plants has the potential to generate broad resistance in plants.

R gene expression induced by a type-III effector triggers disease resistance in rice.

Disease resistance (R) genes in plants encode products that specifically recognise incompatible pathogens and trigger a cascade of events leading to disease resistance in the host plant. R-gene specificity is dictated by both host R genes and cognate avirulence (avr) genes in pathogens. However, the basis of gene-for-gene specificity is not well understood. Here, we report the cloning of the R gene Xa27 from rice and the cognate avr gene avrXa27 from Xanthomonas oryzae pv. oryzae. Resistant and susceptible alleles of Xa27 encode identical proteins. However, expression of only the resistant allele occurs when a rice plant is challenged by bacteria harbouring avrXa27, whose product is a nuclear localized type-III effector. Induction of Xa27 occurs only in the immediate vicinity of infected tissue, whereas ectopic expression of Xa27 resulted in resistance to otherwise compatible strains of the pathogen. Thus Xa27 specificity towards incompatible pathogens involves the differential expression of the R gene in the presence of the AvrXa27 effector.

Development of new transformation-competent artificial chromosome vectors and rice genomic libraries for efficient gene cloning.

The transformation-competent artificial chromosome vector (TAC) system has been shown to be very useful for efficient gene isolation in Arabidopsis thaliana (Proc. Natl. Acad. Sci. USA 96 (1998) 6535). To adapt the vector system for gene isolation in crops, two new TAC vectors and rice genomic libraries were developed. The new vectors pYLTAC17 and pYLTAC27 use the Bar gene and Hpt gene driven by the rice Act1 promoter as the plant selectable markers, respectively, and are suitable for transformation of rice and other grasses. Two representative genomic libraries (I and II) of an Indica rice variety Minghui63, a fertility restorer line for hybrid rice, were constructed with pYLTAC17 using different size classes of partially digested DNA fragments. Library I and library II consisted of 34,560 and 1.2 x 10(5) clones, with average insert sizes of approximately 77 and 39 kb, respectively. The genome coverage of the libraries I and II was estimated to be about 5 and 11 haploid genome equivalents, respectively. Clones of the library I were stored individually in ninety 384-well plates, and those of the library II were collected as bulked pools each containing 30-50 clones and stored in eight 384-well plates. A number of probes were used to hybridize high-density colony filters of the library I prepared by an improved replicating method and each detected 2-9 positive clones. A method for rapid screening of the library II by pooled colony hybridization was developed. A TAC clone having an 80 kb rice DNA insert was successfully transferred into rice genome via Agrobacterium-mediated transformation. The new vectors and the genomic libraries should be useful for gene cloning and genetic engineering in rice and other crops.