Transgenic rice plants expressing the snowdrop lectin gene (<Emphasis Type="Italic">gna</Emphasis>) exhibit high-level resistance to the whitebacked planthopper (<Emphasis Type="Italic">Sogatella furcifera</Emphasis>)

Transgenic rice plants, expressing snowdrop lectin [Galanthus nivalis agglutinin (GNA)], obtained by Agrobacterium-mediated genetic transformation, were evaluated for resistance against the insect, the whitebacked planthopper (WBPH). The transgene gna was driven by the phloem-specific, rice-sucrose synthase promoter RSs1, and the bar was driven by the CaMV 35S promoter. In our previous study, the transgenic status of these lines was confirmed by Southern, Northern and Western blot analyses. Both the transgenes, gna and bar, were stably inherited and co-segregated into progenies in T₁ to T₅ generations. Insect bioassays on transgenic plants revealed the potent entomotoxic effects of GNA on the WBPH. Also, significant decreases were observed in the survival, development and fecundity of the insects fed on transgenic plants. Furthermore, intact GNA was detected in the total proteins of WBPHs fed on these plants. Western blot analysis revealed stable and consistent expression of GNA throughout the growth and development of transgenic plants. Transgenic lines expressing GNA exhibited high-level resistance against the WBPH. As reported earlier, these transgenics also showed substantial resistance against the brown planthopper and green leafhopper .     

Expression of multiple insecticidal genes confers broad resistance against a range of different rice pests

We report the simultaneous introduction of three insecticidal genes (the Bt genes cry1Ac and cry2A, and the snowdrop lectin gene gna) into commercially important indica rice varieties M7 and Basmati 370, by particle bombardment. Transgenic plants expressed Cry1Ac, Cry2A and GNA at different levels, either singly or in combination at 0.03–1%, 0.01–0.5% and 0.01–2.5% of total soluble protein, respectively. The transgenes showed stable transmission and expression, and R₁ transgenic plants provided significant (p<0.01) protection against three of the most important insect pests of rice: rice leaf folder (Cnaphalocrocis medinalis), yellow stemborer (Scirpophaga incertulas) and brown planthopper (Nilaparvata lugens). The triple transformants showed significantly (p<0.05) higher resistance to these insects than plants expressing single transgenes. Bioassays using the triple-transgenic plants showed 100% eradication of the rice leaf folder and yellow stem borer, and 25% reduction in the survival of the brown planthopper. The greatest reduction in insect survival, and the greatest reduction in plant damage, occurred in plants expressing all three transgenes. This approach maximises the utility of gene transfer technology to introduce combinations of genes whose products disrupt different biochemical or physiological processes in the same insect, providing a multi-mechanism defence.     

Development of transgenic rice pure lines with enhanced resistance to rice brown planthopper

Summary Mature seed-derived callus from an elite Chinese japonica rice cv. Ewan 5 was cotransformed with two plasmids, pWRG1515 and pRSSGNAl, containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β-glucuronidase gene (gusA) and the snowdrop (Galanthus nivalis) lectin gene (gna) via particle bombardment. Thirty-five independent transgenic rice plants were regenerated from 177 bombarded calluses. Eighty-three percent of the transgenic plants contained all three genes, as revealed by Southern blot analysis. Western blot analysis revealed that 23 out of 29 gna-containing transgenic plants expressed Galanthus nivalis agglutinin (GNA) (79%) at various levels, with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of all three transgenes (gna, hpt and gusA) in the R2 progeny. Amongst the R2 generation two independent homozygous lines were identified that expressed all three transgenes. Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to rice brown planthopper (Nilaparvata lugens, BPH) by decreasing the survival, overall fecundity of BPH, retarding development, and decreasing the feeding of BPH. These BPH-resistant lines have been incorporated into a rice insect resistance breeding program. This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis-based selection, conferred enhanced resistance to BPH.     

Enhanced insect resistance in Thai rice varieties generated by particle bombardment

We used particle bombardment to transform two elite Thai rice varieties, Khao Dawk Mali 105 (KDML105) and Supanburi 60 (SP60), with the snowdrop lectin gene gna (Galanthus nivalis agglutinin). This gene confers resistance to sap-sucking insects such as the brown planthopper (BPH; Nilaparvata lugens), which is one of the most damaging pests of rice. Traditionally, KDML105 and SP60 have been regarded as recalcitrant to transformation, and this is the first account of successful gene transfer to these varieties. By molecular analysis, we confirmed the recovery of over thirty gna-transgenic lines. GNA protein expression was characterised by western blot analysis, and we achieved expression levels of up to 0.25% total soluble protein. GNA-producing R₁ transgenic plants were significantly more resistant to BPH than control plants (P<0.0001), with 37% and 42% reduction in nymphal survival for constitutive and phloem-specific expression, respectively. Transferring the gna gene to these superior rice varieties thus represents a major step forward for crop improvement in Thailand, and should help to reduce the damage caused by rice pests, and hence increase yields for this vital domestic and export market.     

Mapping and Marker-assisted Selection of a Brown Planthopper Resistance Gene bph2 in Rice (Oryza sativa L.)

Nilaparvata lugens Stål (brown planthopper, BPH), is one of the major insect pests of rice (Oryza sativa L.) in the temperate rice-growing region. In this study, ASD7 harboring a BPH resistance gene bph2 was crossed to a susceptible cultivar C418, a japonica restorer line. BPH resistance was evaluated using 134 F_2:3 lines derived from the cross between “ASD7” and “C418”. SSR assay and linkage analysis were carried out to detect bph2. As a result, the resistant gene bph2 in ASD7 was successfully mapped between RM7102 and RM463 on the long arm of chromosome 12, with distances of 7.6 cM and 7.2 cM, respectively. Meanwhile, both phenotypic selection and marker-assisted selection (MAS) were conducted in the BC₁F₁ and BC₂F₁ populations. Selection efficiencies of RM7102 and RM463 were determined to be 89.9% and 91.2%, respectively. It would be very beneficial for BPH resistance improvement by using MAS of this gene.     

Ecological Trade-offs between Migration and Reproduction Are Mediated by the Nutrition-Sensitive Insulin-Signaling Pathway

Crowding and changes in food availability are two critical environmental conditions that impact an animal''s trajectory toward either migration or reproduction. Many insects facing this challenge have evolved wing polyphenisms. When conditions favor reproduction, wing polyphenic species produce adults that either have no wings or short, non-functional wings. Facultative wing growth reflects a physiological and evolutionary trade-off between migration and reproduction, triggered by environmental conditions. How environmental cues are transduced to produce these alternative forms, and their associated ecological shift from migration to reproduction, remains an important unsolved problem in evolutionary ecology. The brown planthopper, a wing polymorphic insect exhibiting strong trade-offs in investment between migration and reproduction, is one of the most serious rice pests in Asia. In this study, we investigated the function of four genes in the insulin-signaling pathway known to couple nutrition with growth, PI3 Kinase (PI3K), PDK1, Akt (Protein Kinase B), and the forkhead gene FOXO. Using a combination of RNA interference and pharmacological inhibitor treatment, we show that all four genes contribute to tissue level regulation of wing polymorphic development in this insect. As predicted, silencing of the NlPI3K, NlAkt and NlPDK1 through dsRNA and with the pharmacological inhibitor Perifosine resulted in short-winged brown planthoppers, whereas knockdown of NlFOXO resulted in long-winged planthoppers. Morphometric analyses confirm that phenotypes from our manipulations mimic what would be found in nature, i.e., major parameters such as bristle number, wing area and body weight are not significantly different from non-experimental animals. Taken together, these data implicate the insulin-signaling pathway in the transduction of environmental factors into condition-dependent patterns of wing growth in insects.     

Mapping and analysis of a novel candidate Fusarium wilt resistance gene FOC1 in Brassica oleracea

Background

Cabbage Fusarium wilt is a major disease worldwide that can cause severe yield loss in cabbage (Brassica olerecea). Although markers linked to the resistance gene FOC1 have been identified, no candidate gene for it has been determined so far. In this study, we report the fine mapping and analysis of a candidate gene for FOC1 using a double haploid (DH) population with 160 lines and a F₂ population of 4000 individuals derived from the same parental lines.

Results

We confirmed that the resistance to Fusarium wilt was controlled by a single dominant gene based on the resistance segregation ratio of the two populations. Using InDel primers designed from whole-genome re-sequencing data for the two parental lines (the resistant inbred-line 99–77 and the highly susceptible line 99–91) and the DH population, we mapped the resistance gene to a 382-kb genomic region on chromosome C06. Using the F₂ population, we narrowed the region to an 84-kb interval that harbored ten genes, including four probable resistance genes (R genes): Bol037156, Bol037157, Bol037158 and Bol037161 according to the gene annotations from BRAD, the genomic database for B. oleracea. After correcting the model of the these genes, we re-predicted two R genes in the target region: re-Bol037156 and re-Bol0371578. The latter was excluded after we compared the two genes’ sequences between ten resistant materials and ten susceptible materials. For re-Bol037156, we found high identity among the sequences of the resistant lines, while among the susceptible lines, there were two types of InDels (a 1-bp insertion and a 10-bp deletion), each of which caused a frameshift and terminating mutation in the cDNA sequences. Further sequence analysis of the two InDel loci from 80 lines (40 resistant and 40 susceptible) also showed that all 40 R lines had no InDel mutation while 39 out of 40 S lines matched the two types of loci. Thus re-Bol037156 was identified as a likely candidate gene for FOC1 in cabbage.

Conclusions

This work may lay the foundation for marker-assisted selection as well as for further function analysis of the FOC1 gene.     

High-resolution mapping of two rice brown planthopper resistance genes, Bph20(t) and Bph21(t), originating from Oryza minuta

Brown planthopper (BPH) is one of the most destructive insect pests of rice. Wild species of rice are a valuable source of resistance genes for developing resistant cultivars. A molecular marker-based genetic analysis of BPH resistance was conducted using an F₂ population derived from a cross between an introgression line, ‘IR71033-121-15’, from Oryza minuta (Accession number 101141) and a susceptible Korean japonica variety, ‘Junambyeo’. Resistance to BPH (biotype 1) was evaluated using 190 F₃ families. Two major quantitative trait loci (QTLs) and two significant digenic epistatic interactions between marker intervals were identified for BPH resistance. One QTL was mapped to 193.4-kb region located on the short arm of chromosome 4, and the other QTL was mapped to a 194.0-kb region on the long arm of chromosome 12. The two QTLs additively increased the resistance to BPH. Markers co-segregating with the two resistance QTLs were developed at each locus. Comparing the physical map positions of the two QTLs with previously reported BPH resistance genes, we conclude that these major QTLs are new BPH resistance loci and have designated them as Bph20(t) on chromosome 4 and Bph21(t) on chromosome 12. This is the first report of BPH resistance genes from the wild species O. minuta. These two new genes and markers reported here will be useful to rice breeding programs interested in new sources of BPH resistance.     

Genetic mapping of the rice resistance-breaking gene of the brown planthopper Nilaparvata lugens

Host plant resistance has been widely used for controlling the major rice pest brown planthopper (BPH, Nilaparvata lugens). However, adaptation of the wild BPH population to resistance limits the effective use of resistant rice varieties. Quantitative trait locus (QTL) analysis was conducted to identify resistance-breaking genes against the anti-feeding mechanism mediated by the rice resistance gene Bph1. QTL analysis in iso-female BPH lines with single-nucleotide polymorphism (SNP) markers detected a single region on the 10th linkage group responsible for the virulence. The QTL explained from 57 to 84% of the total phenotypic variation. Bulked segregant analysis with next-generation sequencing in F₂ progenies identified five SNPs genetically linked to the virulence. These analyses showed that virulence to Bph1 was controlled by a single recessive gene. In contrast to previous studies, the gene-for-gene relationship between the major resistance gene Bph1 and virulence gene of BPH was confirmed. Identified markers are available for map-based cloning of the major gene controlling BPH virulence to rice resistance.     

Development of 25 near-isogenic lines (NILs) with ten BPH resistance genes in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.): production,resistance spectrum,and molecular analysis

Key message

A first set of 25 NILs carrying ten BPH resistance genes and their pyramids was developed in the background of indica variety IR24 for insect resistance breeding in rice.

Abstract

Brown planthopper (Nilaparvata lugens Stal.) is one of the most destructive insect pests in rice. Development of near-isogenic lines (NILs) is an important strategy for genetic analysis of brown planthopper (BPH) resistance (R) genes and their deployment against diverse BPH populations. A set of 25 NILs with 9 single R genes and 16 multiple R gene combinations consisting of 11 two-gene pyramids and 5 three-gene pyramids in the genetic background of the susceptible indica rice cultivar IR24 was developed through marker-assisted selection. The linked DNA markers for each of the R genes were used for foreground selection and confirming the introgressed regions of the BPH R genes. Modified seed box screening and feeding rate of BPH were used to evaluate the spectrum of resistance. BPH reaction of each of the NILs carrying different single genes was variable at the antibiosis level with the four BPH populations of the Philippines. The NILs with two- to three-pyramided genes showed a stronger level of antibiosis (49.3–99.0%) against BPH populations compared with NILs with a single R gene NILs (42.0–83.5%) and IR24 (10.0%). Background genotyping by high-density SNPs markers revealed that most of the chromosome regions of the NILs (BC₃F₅) had IR24 genome recovery of 82.0–94.2%. Six major agronomic data of the NILs showed a phenotypically comparable agronomic performance with IR24. These newly developed NILs will be useful as new genetic resources for BPH resistance breeding and are valuable sources of genes in monitoring against the emerging BPH biotypes in different rice-growing countries.

     

Inheritance of resistance to Bacillus thuringiensis Cry1Ab protein in the sugarcane borer (Lepidoptera: Crambidae)

Inheritance traits of a Cry1Ab-resistant strain of the sugarcane borer, Diatraea saccharalis (F.) were analyzed using various genetic crosses. Reciprocal parental crosses between Cry1Ab-susceptible and Cry1Ab-resistant populations, F₁ by F₁ crosses, and backcrosses of F₁ with the Cry1Ab-resistant population were successfully completed. Larval mortality of the parental and cross-populations were assayed on Cry1Ab diet and Bacillus thuringiensis (Bt)-corn leaf tissue. Maternal effects and sex linkage were examined by comparing the larval mortality between the two F₁ populations. Dominance levels of resistance were measured by comparing the larval mortality of the Cry1Ab-resistant, -susceptible, and -heterozygous populations. Number of genes associated with the resistance was evaluated by fitting the observed mortality of F₂ and backcross populations with a Mendelian monogenic inheritance model. Cry1Ab resistance in D. saccharalis was likely inherited as a single or a few tightly linked autosomal genes. The resistance was incompletely recessive on Bt corn leaf tissue, while the effective dominance levels (D_ML) of resistance increased as Cry1Ab concentrations decreased with Cry1Ab-treated diet. D_ML estimated based on larval mortality on intact Bt corn plants reported in a previous study ranged from 0.08 to 0.26. This variability in D_ML levels of Cry1Ab resistance in D. saccharalis suggests that Bt corn hybrids must express a sufficient dose of Bt proteins to make the resistance genes functionally recessive. Thus, Bt resistant heterozygous individuals can be killed as desired in the “high/dose refuge” resistance management strategy for Bt corn.     

High-resolution mapping of the brown planthopper resistance gene Bph6 in rice and characterizing its resistance in the 9311 and Nipponbare near isogenic backgrounds

Brown planthopper (Nilaparvata lugens Stål, BPH) is one of the most destructive insect pests of rice. Exploring resistance genes from diverse germplasms and incorporating them into cultivated varieties are critical for controlling this insect. The rice variety Swarnalata was reported to carry a resistance gene (designated Bph6), which has not yet been assigned to a chromosome location and the resistance mechanism is still unknown. In this study, we identified and mapped this gene using the F₂ and backcrossing populations and characterized its resistance in indica 9311 and japonica Nipponbare using near isogenic lines (NILs). In analysis of 9311/Swarnalata F₂ population, the Bph6 gene was located on the long arm of chromosome 4 between the SSR markers RM6997 and RM5742. The gene was further mapped precisely to a 25-kb region delimited between the STS markers Y19 and Y9; and the distance between these markers is 25-kb in Nipponbare genome. The Bph6 explained 77.5% of the phenotypic variance of BPH resistance in F₂ population and 84.9% in BC₂F₂ population. Allele from Swarnalata significantly increased resistance to the BPH, resulted in a reduced damage score. In characterization of Bph6-mediated resistance, the BPH insects showed significant preference between NIL-9311 and 9311 in 3 h and between NIL-NIP and Nipponbare in 120 h after release. BPH growth and development were inhibited, and the insect’s survival rates were lower on Bph6-NIL plants, compared with the parents 9311 and Nipponbare. The results indicate that the Bph6 exerted prolonged antixenotic and antibiotic effects in Bph6-NIL plants, and NIL-9311 plants showed a quicker and stronger effect toward BPH than NIL-NIP plants.     

Transgenic rice expressing Allium sativum leaf lectin with enhanced resistance against sap-sucking insect pests

Mannose binding Allium sativum leaf agglutinin (ASAL) has been shown to be antifeedant and insecticidal against sap-sucking insects. In the present investigation, ASAL coding sequence was expressed under the control of CaMV35S promoter in a chimeric gene cassette containing plant selection marker, hpt and gusA reporter gene of pCAMBIA1301 binary vector in an elite indica rice cv. IR64. Many fertile transgenic plants were generated using scutellar calli as initial explants through Agrobacterium-mediated transformation technology. GUS activity was observed in selected calli and in mature plants. Transformation frequency was calculated to be ~12.1%±0.351 (mean ± SE). Southern blot analyses revealed the integration of ASAL gene into rice genome with a predominant single copy insertion. Transgene localization was detected on chromosomes of transformed plants using PRINS and C-PRINS techniques. Northern and western blot analyses determined the expression of transgene in transformed lines. ELISA analyses estimated ASAL expression up to 0.72 and 0.67% of total soluble protein in T₀ and T₁ plants, respectively. Survival and fecundity of brown planthopper and green leafhopper were reduced to 36% (P<0.01), 32% (P<0.05) and 40.5, 29.5% (P<0.001), respectively, when tested on selected plants in comparison to control plants. Specific binding of expressed ASAL to receptor proteins of insect gut was analysed. Analysis of T₁ progenies confirmed the inheritance of the transgenes. Thus, ASAL promises to be a potential component in insect resistance rice breeding programme.     

Production of Transgenic Rice Homozygous Lines with Enhanced Resistance to the Rice Brown Planthopper

Mature seed‐derived callus from an elite Chinese japonica rice (Oryza sativa L.) cv. Eyi 105 was cotransformed with two plasmids, pWRG1515 and pRSSGNA1,containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β‐glucuronidase gene (gusA) and the snow‐drop (Galanthus nivalis) lectin gene (gna) via particle bombardment. After two rounds of selection on hygromycin‐containing medium, resistant callus was transferred to hygromycin‐containing regeneration medium for plant regeneration. Twenty‐six independent transgenic rice plants were regenerated from 152 bombarded calli with a transformation frequency of 17%. Seventy‐three percent of transgenic plants contained all three genes, which was revealed by PCR/Southern blot analysis. Thirteen out of 19 transgenic plants containing the gna gene expressed GNA (68%) at various levels with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parentplants showing 3:1 Mendelian segregation patterns, we identified three independent homozygous lines containing and expressing all three transgenes.Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to the rice brown planthopper (Nilaparvata lugens, BPH) by decreasing BPH survival and overall fecundity, retarding BPH development and reducing BPH feeding.This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis‐based selection, conferred enhanced resistance to BPH, one of the most damaging insect pests in rice.     

Mapping and pyramiding of two major genes for resistance to the brown planthopper (<Emphasis Type="Italic">Nilaparvata lugens</Emphasis> [Stål]) in the rice cultivar ADR52

The brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the most serious and destructive pests of rice, and can be found throughout the rice-growing areas of Asia. To date, more than 24 major BPH-resistance genes have been reported in several Oryza sativa ssp. indica cultivars and wild relatives. Here, we report the genetic basis of the high level of BPH resistance derived from an Indian rice cultivar, ADR52, which was previously identified as resistant to the whitebacked planthopper (Sogatella furcifera [Horváth]). An F₂ population derived from a cross between ADR52 and a susceptible cultivar, Taichung 65 (T65), was used for quantitative trait locus (QTL) analysis. Antibiosis testing showed that multiple loci controlled the high level of BPH resistance in this F₂ population. Further linkage analysis using backcross populations resulted in the identification of BPH-resistance (antibiosis) gene loci from ADR52. BPH25 co-segregated with marker S00310 on the distal end of the short arm of chromosome 6, and BPH26 co-segregated with marker RM5479 on the long arm of chromosome 12. To characterize the virulence of the most recently migrated BPH strain in Japan, preliminary near-isogenic lines (pre-NILs) and a preliminary pyramided line (pre-PYL) carrying BPH25 and BPH26 were evaluated. Although both pre-NILs were susceptible to the virulent BPH strain, the pre-PYL exhibited a high level of resistance. The pyramiding of resistance genes is therefore likely to be effective for increasing the durability of resistance against the new virulent BPH strain in Japan.     

Marker-assisted pyramiding of eight QTLs/genes for seven different traits in common wheat (Triticum aestivum L.)

Common wheat (Triticum aestivum L.) contributes substantially to global food and nutritional security. Thus, an important goal of wheat breeding is to develop high-yielding varieties with better nutritional quality and resistance to all major diseases. During the present study, in the background of a popular elite wheat cultivar PBW343, we pyramided eight quantitative trait loci (QTLs)/genes for four grain quality traits (high grain weight, high grain protein content, pre-harvest sprouting tolerance, and desirable high-molecular-weight glutenin subunits) and resistance against the three rusts. For pyramiding eight QTLs/genes, four improved PBW343 lines, each carrying different combinations of the desired QTLs/genes (developed by us earlier), were crossed in pairs to produce two single-cross F₁ hybrids. The single-cross F₁ hybrids were intercrossed to produce a double-cross hybrid (DCH). Using marker-assisted selection in five consecutive generations (DCHF₁–DCHF₅), four pyramided lines (PYLs) were selected, each with all the eight desired QTLs/genes in homozygous state. The phenotypic characterization of the progenies of these PYLs suggested that the genetic background of PBW343 was retained in all these four PYLs. Therefore, these PYLs should prove useful in future wheat breeding programs for improving not only the grain quality, but also the durability of resistance against all three rusts. Multi-year/multi-location trials are planned for these pyramided lines to evaluate their potential for release as a next-generation improved version of wheat cv. PBW343 for commercial cultivation.     

Fine mapping and characterization of BPH27, a brown planthopper resistance gene from wild rice (Oryza rufipogon Griff.)

The brown planthopper (Nilaparvata lugens Stål; BPH) is one of the most serious rice pests worldwide. Growing resistant varieties is the most effective way to manage this insect, and wild rice species are a valuable source of resistance genes for developing resistant cultivars. BPH27 derived from an accession of Guangxi wild rice, Oryza rufipogon Griff. (Accession no. 2183, hereafter named GX2183), was primarily mapped to a 17-cM region on the long arm of the chromosome four. In this study, fine mapping of BPH27 was conducted using two BC₁F₂ populations derived from introgression lines of GX2183. Insect resistance was evaluated in the BC₁F₂ populations with 6,010 individual offsprings, and 346 resistance extremes were obtained and employed for fine mapping of BPH27. High-resolution linkage analysis defined the BPH27 locus to an 86.3-kb region in Nipponbare. Regarding the sequence information of rice cultivars, Nipponbare and 93-11, all predicted open reading frames (ORFs) in the fine-mapping region have been annotated as 11 types of proteins, and three ORFs encode disease-related proteins. Moreover, the average BPH numbers showed significant differences in 96–120 h after release in comparisons between the preliminary near-isogenic lines (pre-NILs, lines harboring resistance genes) and BaiR54. BPH growth and development were inhibited and survival rates were lower in the pre-NIL plants compared with the recurrent parent BaiR54. The pre-NIL exhibited 50.7 % reductions in population growth rates (PGR) compared to BaiR54. The new development in fine mapping of BPH27 will facilitate the efforts to clone this important resistant gene and to use it in BPH-resistance rice breeding.     

Differential expression of vitellogenin mRNA and protein in response to rice resistance genes in two strains of <Emphasis Type="Italic">Nilaparvata</Emphasis><Emphasis Type="Italic">lugens</Emphasis> (Hemiptera: Delphacidae) with different levels of virulence

Controlling the brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is a difficult task in rice (Oryza sativa L.) production. We focused on vitellogenins (Vg), which are the major yolk protein precursors of vitellins and play an important role in the reproduction of oviparous species, including insects. We studied the accumulation of Vg mRNA and protein in a virulent BPH strain, Nagasaki-03, and a nonvirulent strain, Hatano-66, after rearing them on four rice lines. The rice lines used were two single resistance gene introgression lines, Norin-PL3 (Bph1 carrier) and Norin-PL4 (bph2 carrier), a pyramided line in which both genes were combined, and a susceptible japonica recurrent parent Tsukushibare. RT-PCR and quantitative RT-PCR analyses showed that the Vg mRNA level decreased greatly in Hatano-66 on the resistant lines. In contrast, the level of reduction on the resistant lines was much less in Nagasaki-03. Immunoblot analysis showed that Nagasaki-03 retained comparable levels of 175 kDa Vg protein on both the susceptible and resistant lines, whereas in Hatano-66, no Vg protein was detected on the resistant lines. Our results showed that BPH resistance genes caused differential reduction in the accumulation of Vg mRNA and protein, leading to the retardation of BPH reproduction on the resistant host rice plants.