Resistance to green leafhopper (Nephotettix virescens) and brown planthopper (Nilaparvata lugens) in transgenic rice expressing snowdrop lectin (Galanthus nivalis agglutinin; GNA)

Transgenic rice plants expressing snowdrop lectin (Galanthus nivalis agglutinin; GNA) were screened for resistance to green leafhopper (Nephotettix virescens; GLH), a major homopteran pest of rice. Survival was reduced by 29% and 53% (P<0.05) respectively, on plants where GNA expression was tissue-specific (phloem and epidermal layer) or constitutive. Similar levels of resistance in GNA-expressing transgenic rice were previously reported for rice brown planthopper (Nilaparvata lugens; BPH). GNA binding to glycoproteins in gut tissues showed that BPH contained more "receptors" than GLH, and that the binding affinity was stronger, particularly in the midgut. Subsequent toxicity of GNA is thus unlikely to be directly related to the amount of lectin bound. GNA was not detected in the honeydew of either insect species when they were fed on GNA-expressing plants, in contrast to results from artificial diet studies. This result suggests that GNA is not being delivered to the insect efficiently. When offered a free choice vs control plants, BPH nymphs tended to avoid plants expressing GNA; avoidance was less pronounced and took longer to develop on plants where GNA expression was tissue-specific, In contrast to BPH, GLH nymphs were attracted to plants expressing GNA, whether constitutively or in a tissue-specific manner.     

Identification of a new locus, Ptr(t), required for rice blast resistance gene Pi-ta-mediated resistance

Resistance to the blast pathogen Magnaporthe oryzae is proposed to be initiated by physical binding of a putative cytoplasmic receptor encoded by a nucleotide binding site-type resistance gene, Pi-ta, to the processed elicitor encoded by the corresponding avirulence gene AVR-Pita. Here, we report the identification of a new locus, Ptr(t), that is required for Pi-ta-mediated signal recognition. A Pi-ta-expressing susceptible mutant was identified using a genetic screen. Putative mutations at Ptr(t) do not alter recognition specificity to another resistance gene, Pi-k(s), in the Pi-ta homozygote, indicating that Ptr(t) is more likely specific to Pi-ta-mediated signal recognition. Genetic crosses of Pi-ta Ptr(t) and Pi-ta ptr(t) homozygotes suggest that Ptr(t) segregates as a single dominant nuclear gene. A ratio of 1:1 (resistant/susceptible) of a population of BC1 of Pi-ta Ptr(t) with pi-ta ptr(t) homozygotes indicates that Pi-ta and Ptr(t) are linked and cosegregate. Genotyping of mutants of pi-ta ptr(t) and Pi-ta Ptr(t) homozygotes using ten simple sequence repeat markers at the Pi-ta region determined that Pi-ta and Ptr(t) are located within a 9-megabase region and are of indica origin. Identification of Ptr(t) is a significant advancement in studying Pi-ta-mediated signal recognition and transduction.     

Molecular mapping of a novel gene, Grh5, conferring resistance to green rice leafhopper (Nephotettix cincticeps Uhler) in rice, Oryza sativa L.

The green rice leafhopper (GRH), Nephotettix cincticeps Uhler, is one of the most serious insect pests affecting cultivated rice (Oryza sativa L.) in temperate regions of East Asia. An accession of the wild rice species, Oryza rufipogon Griff. (W1962), was found to be highly resistant to GRH by an antibiosis test. To understand the genetic basis of the GRH resistance, a BC₁F₁ population derived from a cross between a susceptible Japonica variety, Taichung 65 (T65), and a highly resistant accession W1962 was analyzed by quantitative trait loci (QTL) mapping. A single major QTL for GRH resistance was detected on rice chromosome 8. A nearly isogenic population containing segments of the targeted QTL region derived from W1962 was then developed through advanced backcrossing with marker-assisted selection. Further molecular mapping using a BC₄F₂ population revealed that a new resistance gene, designated as Green rice leafhopper resistance 5 (Grh5), was located on the distal region of the long arm of chromosome 8 and tightly linked to the simple sequence repeat markers RM3754 and RM3761. A nearly isogenic line (NIL) carrying Grh5 was subsequently developed in the progeny of the mapping population. The resistance level of Grh5-NIL was compared with those of developed NILs for GRH resistance and was found to have the highest resistance. The DNA markers found to be closely linked to Grh5 would be useful for marker-assisted selection for the improvement of resistance to GRH in rice.     

Life table analysis of the green rice leafhopper,Nephotettix virescens (distant) (Hemiptera: Cicadellidae), and efficient vector of rice tungro disease in asynchronous rice fields in Indonesia

Population dynamics of Nephotettix virescens was studied in 17 paddy fields transplanted at intervals of about 1 month in 1988–1990. The adult density was highest either in the immigrant or the 1st generation and sharply decreased to the 2nd generation. The survival rate of the 1st generation was lowest in the transition season when areal population density increased. Key factor analysis revealed that the nymphal and adult mortality of the 1st generation (kn) was the principal source of population fluctuations. No significant correaltion was found between kn and natural enemy density, natural enemy density/healthy egg density, or the precipitation during the nymphal period. On these bases adult emigration was suspected to be the key factor. Areal population build-up of N. virescens in the transition season was considered to occur as a result of increasing immigration to young stages of rice.     

Population dynamics of the green leafhopper,Nephotettix virescens Distant (Hemiptera: Cicadellidae) in synchronized and staggered transplanting areas of paddy fileds in Indonesia

The population dynamics of Nephotettix virescens, a vector of rice tungro virus disease was investigated in a synchronized transplanting area at Jatisari (1984–1986), West Java and in a staggered transplanting area at Sidan (1986–1988), Bali, Indonesia. The FARMCOP suction sampler was employed for population censuses of N. virescens and its natural enemies. The population growth pattern was affected by transplanting pattern: In the staggered transplanting area, the population density increased from the immigrant generation to the first generation, and sharply decrease thereafter, while in the synchronized transplanting area the population density often reached the highest peak in the second generation. The degree of contageousness in the spatial distribution of N. virescens was negatively correlated with population density of the immigrant generation.     

Genetic and physical mapping of a new gene for bacterial blight resistance in rice

The inheritance of resistance for bacterial blight, caused by Xanthomonas oryzae pv. oryzae ( Xoo), was studied in Minghui 63, an elite restorer line for a number of widely used rice hybrids in China. A new dominant gene against a Chinese Xoo strain JL691 in both the seedling and adult stages was identified in Minghui 63 and designated as Xa26( t). Using a total of 477 highly susceptible individuals from an F(2) population, the Xa26( t) locus was mapped to a region of about 1.68 cM. This locus co-segregated with marker R1506 and was 0.21 cM from marker RM224 on one side and 1.47 cM from marker Y6855RA on the other side, in rice chromosome 11. A contig map, composed of five non-redundant bacterial artificial chromosome (BAC) clones and spanning approximately 500 kb in length, was constructed. Analysis of recombination events in the Xa26( t) region with the highly susceptible F(2) individuals anchored the gene locus to a region covered by three overlapped BAC clones. Assay of the lines showing a double crossover in marker loci flanking Xa26( t), in a population of recombinant inbred lines carrying Xa26( t), further delineated the gene to a 20-kb fragment. The Xa26( t) locus is tightly linked to another bacterial blight resistance gene locus, Xa4.     

A multiple gene complex on rice chromosome 4 is involved in durable resistance to rice blast

Quantitative trait loci (QTLs) for resistance to rice blast offer a potential source of durable disease resistance in rice. However, few QTLs have been validated in progeny testing, on account of their small phenotypic effects. To understand the genetic basis for QTL-mediated resistance to blast, we dissected a resistance QTL, qBR4-2, using advanced backcross progeny derived from a chromosome segment substitution line in which a 30- to 34-Mb region of chromosome 4 from the resistant cultivar Owarihatamochi was substituted into the genetic background of the highly susceptible Aichiasahi. The analysis resolved qBR4-2 into three loci, designated qBR4-2a, qBR4-2b, and qBR4-2c. The sequences of qBR4-2a and qBR4-2b, which lie 181 kb apart from each other and measure, 113 and 32 kb, respectively, appear to encode proteins with a putative nucleotide-binding site (NBS) and leucine-rich repeats (LRRs). Sequence analysis of the donor allele of qBR4-2a, the region with the largest effect among the three, revealed sequence variations in the NBS-LRR region. The effect of qBR4-2c was smallest among the three, but its combination with the donor alleles of qBR4-2a and qBR4-2b significantly enhanced blast resistance. qBR4-2 comprises three tightly linked QTLs that control blast resistance in a complex manner, and thus gene pyramiding or haplotype selection is the recommended strategy for improving QTL-mediated resistance to blast disease through the use of this chromosomal region.     

A telomeric avirulence gene determines efficacy for the rice blast resistance gene Pi-ta

Genetic mapping showed that the rice blast avirulence gene AVR-Pita is tightly linked to a telomere on chromosome 3 in the plant pathogenic fungus Magnaporthe grisea. AVR-Pita corresponds in gene-for-gene fashion to the disease resistance (R) gene Pi-ta. Analysis of spontaneous avr-pita(-) mutants indicated that the gene is located in a telomeric 6.5-kb BglII restriction fragment. Cloning and DNA sequencing led to the identification of a candidate gene with features typical of metalloproteases. This gene is located entirely within the most distal 1.5 kb of the chromosome. When introduced into virulent rice pathogens, the cloned gene specifically confers avirulence toward rice cultivars that contain Pi-ta. Frequent spontaneous loss of AVR-Pita appears to be the result of its telomeric location. Diverse mutations in AVR-Pita, including point mutations, insertions, and deletions, permit the fungus to avoid triggering resistance responses mediated by Pi-ta. A point mutation in the protease consensus sequence abolishes the AVR-Pita avirulence function.     

Feeding strategy for the production of the new antibiotic myxovirescin A from Myxococcus virescens (Myxobacterales)

Summary Myxovirescin A is a new antibiotic from Myxococcus virescens. Conditions of growth on peptone media result in the antibiotic being secreted during the transition to the stationary phase. When growth is exponential, no detectable production occurs.In an attempt to improve production of the antibiotic, peptone was fed to the peptone-limited culture at differing feed rates. Product formation was found to be dependent on the peptone supply, and the product concentration could be improved from 0.04 to 2 mg/l myxovirescin A.Article No. 13 on antibiotics from gliding bacteria Article No. 12: Thierbach G, Reichenbach H (1983). The effect of the new antibiotic myxothiazol on the respiration of Paracoccus denitrificans. Arch Microbiol 134: 104–107     

NBS-LRR resistance gene homologues in rice

Twenty three DNA fragments with a size of about 520 bp have been cloned from rice genome by PCR amplification using primers designed according to the conserved region of most plant resistance (R) genes which have Nucleotide Binding Site (NBS) and Leucine-Rich Repeat (LRR) domains. Homologous comparison showed that these fragments contained typical motifs of the NBS-LRR resistance gene class, kinase 1a, kinase 2a, kinase 3a and domain 2. Thus they were named R gene homologous sequences (RS). These RS were divided into 4 groups by clustering analysis and mapped onto chromosomes 1, 3, 4, 7, 8, 9, 10 and 11, respectively, by genetic mapping. Ten RS were located in the chromosomal intervals where known R genes had been mapped. Further RFLP analysis of an RS, RS13, near the bacterial blight resistance gene Xa4 locus on chromosome 11 among near isogenic lines and pyramiding lines of Xa4 showed that RS13 was possibly amplified from the gene family of Xa4.     

NBS-LRR resistance gene homologues in rice

Twenty three DNA fragments with a size of about 520 bp have been cloned from rice genome by PCR amplification using primers designed according to the conserved region of most plant resistance (R) genes which have Nucleotide Binding Site (NBS) and Leucine-Rich Repeat (LRR) domains. Homologous comparison showed that these fragments contained typical motifs of the NBS-LRR resistance gene class, kinase 1a, kinase 2a, kinase 3a and domain 2. Thus they were named R gene homologous sequences (RS). These RS were divided into 4 groups by clustering analysis and mapped onto chromosomes 1, 3, 4, 7, 8, 9, 10 and 11, respectively, by genetic mapping. Ten RS were located in the chromosomal intervals where known R genes had been mapped. Further RFLP analysis of an RS, RS13, near the bacterial blight resistance geneXa4 locus on chromosome 11 among near isogenic lines and pyramiding lines ofXa4 showed that RS13 was possibly amplified from the gene family ofXa4.     

CryIIIA toxin gene expression in transgenic rice confers resistance to rice water weevil

The rice water weevil (RWW), Lissorhoptrus oryzophilus Kuschel, is the most widely distributed and destructive early season insect pest of rice, Oryza sativa L. worldwide. The rice plants were transformed with cryIIIA insecticidal gene as well as with the bar gene coding phosphinothricin acetyltransferase. CryIIIA gene under the control of a modified RCg2 promoter drives the insect-toxic gene expression in roots and/or leaves. The cryIIIA gene was transferred into O. sativa L. cv. Nakdong by Agrobacterium-mediated transformation. Stable integration of the transgene was confirmed in putative transformed rice by Southern blot analysis. The expression of the cryIIIA toxin gene in the roots of transgenic rice plants was verified by RT-PCR and immunoblot analysis. Transgenic rice plants were also evaluated for resistance to natural infestations of the RWW under field conditions between 2007 and 2011. The transgenic Btt8R and Btt12R lines reduced the growth rate of RWW larvae and pupae populations compared with non-transgenic control plants by approximately 52 and 58 %, respectively. To further examine the efficacy of the RWW bioassay, we used pots and performed experiments in trays and under field conditions in 2012. The Btt12R line reduced the total populations of RWW larvae and pupae in trays and under field conditions by 56 and 45 %, respectively. The bioassay experiments conducted over 6 years, showed a significant reduction rate of RWW larvae and pupae populations demonstrating that the cryIIIA gene in transgenic rice confers resistance to RWW.     

High-resolution mapping of a new brown planthopper (BPH) resistance gene, Bph18(t), and marker-assisted selection for BPH resistance in rice (Oryza sativa L.)

Brown planthopper (BPH) is a destructive insect pest of rice in Asia. Identification and the incorporation of new BPH resistance genes into modern rice cultivars are important breeding strategies to control the damage caused by new biotypes of BPH. In this study, a major resistance gene, Bph18(t), has been identified in an introgression line (IR65482-7-216-1-2) that has inherited the gene from the wild species Oryza australiensis. Genetic analysis revealed the dominant nature of the Bph18(t) gene and identified it as non-allelic to another gene, Bph10 that was earlier introgressed from O. australiensis. After linkage analysis using MapMaker followed by single-locus ANOVA on quantitatively expressed resistance levels of the progenies from an F₂ mapping population identified with marker allele types, the Bph18(t) gene was initially located on the subterminal region of the long arm of chromosome 12 flanked by the SSR marker RM463 and the STS marker S15552. The corresponding physical region was identified in the Nipponbare genome pseudomolecule 3 through electronic chromosome landing (e-landing), in which 15 BAC clones covered 1.612 Mb. Eleven DNA markers tagging the BAC clones were used to construct a high-resolution genetic map of the target region. The Bph18(t) locus was further localized within a 0.843-Mb physical interval that includes three BAC clones between the markers R10289S and RM6869 by means of single-locus ANOVA of resistance levels of mapping population and marker-gene association analysis on 86 susceptible F₂ progenies based on six time-point phenotyping. Using gene annotation information of TIGR, a putative resistance gene was identified in the BAC clone OSJNBa0028L05 and the sequence information was used to generate STS marker 7312.T4A. The marker allele of 1,078 bp completely co-segregated with the BPH resistance phenotype. STS marker 7312.T4A was validated using BC₂F₂ progenies derived from two temperate japonica backgrounds. Some 97 resistant BC₂F₂ individuals out of 433 screened completely co-segregated with the resistance-specific marker allele (1,078 bp) in either homozygous or heterozygous state. This further confirmed a major gene-controlled resistance to the BPH biotype of Korea. Identification of Bph18(t) enlarges the BPH resistance gene pool to help develop improved rice cultivars, and the PCR marker (7312.T4A) for the Bph18(t) gene should be readily applicable for marker-assisted selection (MAS). K. K. Jena and J. U. Jeung contributed equally to this study.     

Constitutive and tissue-specific differential expression of the cryIA(b) gene in transgenic rice plants conferring resistance to rice insect pest

 The truncated chimeric Bt gene, cryIA(b) of Bacillus thuringiensis, driven by two constitutive promoters, 35S from CaMV and Actin-1 from rice, and two tissue-specific promoters, pith tissue and pepcarboxylase (PEPC) for green tissue from maize, was introduced into several varieties of rice (indica and japonica) by microprojectile bombardment and protoplast systems. A total of 1800 putative transgenic Bt rice plants could be produced. Southern analysis revealed that more than 100 independently transformed plants could be confirmed for integration of the cryIA(b) gene. High levels of CryIA(b) proteins were obtained in the green tissue (leaves and stem) of many plants using the PEPC promoter. There was little difference in Bt protein level in leaves and stems from transgenic plants with the 35 S or Actin-1 promoter. Out of 800 Southern-positive plants that were bioassayed, 81 transgenic plants showed 100% mortality of insect larvae of the yellow stem borer (Scirpophaga incertulas). The transgene, cryIA(b), driven by different promoters showed a wide range of expression (low to high) of Bt proteins stably inherited in a number of rice varieties with enhanced yellow stem borer resistance. This first report of transgenic indica Bt rice plants with the PEPC or pith promoter either alone or in combination should provide a better strategy for providing rice plants with protection against insect pest resistance, minimizing the expression of the CryIA(b) protein in seeds and other tissues. Received: 12 November 1997 / Accepted: 25 November 1997     

Pi35(t), a new gene conferring partial resistance to leaf blast in the rice cultivar Hokkai 188

The japonica rice cultivar Hokkai 188 shows a high level of partial resistance to leaf blast. For mapping genes conferring the resistance, a set of 190 F₂ progeny/F₃ families was developed from the cross between the indica rice cultivar Danghang-Shali, with a low level of partial resistance, and Hokkai 188. Partial resistance to leaf blast in the F₃ families was assessed in upland nurseries. From a primary microsatellite (SSR) linkage map and QTL analysis using a subset of 126 F₂ progeny/F₃ families randomly selected from the above set, one major QTL located on chromosome 1 was detected in the vicinity of SSR marker RM1216. This QTL was responsible for 69.4% of the phenotypic variation, and Hokkai 188 contributed the resistance allele. Segregation analysis in the F₃ families for partial resistance to leaf blast was in agreement with the existence of a major gene, and the gene was designated as Pi35(t). Another QTL detected on chromosome 8 was minor, explained 13.4% of the phenotypic variation, and an allele of Danghang-Shali increased the level of resistance in this QTL. Additional SSR markers of the targeted Pi35(t) region were further surveyed in the 190 F₂ plants, and Pi35(t) was placed in a 3.5-cM interval flanked by markers RM1216 and RM1003.