Fine-mapping and identification of a novel locus <Emphasis Type="Italic">Rsc15</Emphasis> underlying soybean resistance to <Emphasis Type="Italic">Soybean mosaic virus</Emphasis>

Key message

Rsc15, a novel locus underlying soybean resistance to SMV, was fine mapped to a 95-kb region on chromosome 6. The Rsc15- mediated resistance is likely attributed to the gene GmPEX14 , the relative expression of which was highly correlated with the accumulation of H ₂ O ₂ along with the activities of POD and CAT during the early stages of SMV infection in RN-9.

Abstract

Soybean mosaic virus (SMV) causes severe yield losses and seed quality deterioration in soybean [Glycine max (L.) Merr.] worldwide. A series of single dominant SMV resistance genes have been identified on respective soybean chromosomes 2, 13 and 14, while one novel locus, Rsc15, underlying resistance to the virulent SMV strain SC15 from soybean cultivar RN-9 has been recently mapped to a 14.6-cM region on chromosome 6. However, candidate gene has not yet been identified within this region. In the present study, we aimed to fine map the Rsc15 region and identify candidate gene(s) for this invaluable locus. High-resolution fine-mapping revealed that the Rsc15 gene was located in a 95-kb genomic region which was flanked by the two simple sequence repeat (SSR) markers SSR_06_17 and BARCSOYSSR_06_0835. Allelic sequence comparison and expression profile analysis of candidate genes inferred that the gene Glyma.06g182600 (designated as GmPEX14) was the best candidate gene attributing for the resistance of Rsc15, and that genes encoding receptor-like kinase (RLK) (i.e., Glyma.06g175100 and Glyma.06g184400) and serine/threonine kinase (STK) (i.e., Glyma.06g182900 and Glyma.06g183500) were also potential candidates. High correlations were established between the relative expression level of GmPEX14 and the hydrogen peroxide (H₂O₂) concentration and activities of catalase (CAT) and peroxidase (POD) during the early stages of SMV-SC15 infection in RN-9. The results of the present study will be useful in marker-assisted breeding for SMV resistance and will lead to further understanding of the molecular mechanisms of host resistance against SMV.

     

Mapping of yield-related QTLs in pepper in an interspecific cross of <Emphasis Type="Italic">Capsicum annuum</Emphasis> and <Emphasis Type="Italic">C. frutescens</Emphasis>

An advanced backcross QTL study was performed in pepper using a cross between the cultivated species Capsicum annuum cv. Maor and the wild C. frutescens BG 2816 accession. A genetic map from this cross was constructed, based on 248 BC(2) plants and 92 restriction fragment length polymorphism (RFLP) markers distributed throughout the genome. Ten yield-related traits were analyzed in the BC(2) and BC(2)S(1) generations, and a total of 58 quantitative trait loci (QTLs) were detected; the number of QTLs per trait ranged from two to ten. Most of the QTLs were found in 11 clusters, in which similar QTL positions were identified for multiple traits. Unlike the high percentage of favorable QTL alleles discovered in wild species of tomato and rice, only a few such QTL alleles were detected in BG 2816. For six QTLs (10%), alleles with effects opposite to those expected from the phenotype were detected in the wild species. The use of common RFLP markers in the pepper and tomato maps enabled possible orthologous QTLs in the two species to be determined. The degree of putative QTL orthology for the two main fruit morphology traits-weight and shape-varied considerably. While all eight QTLs identified for fruit weight in this study could be orthologous to tomato fruit weight QTLs, only one out of six fruit shape QTLs found in this study could be orthologous to tomato fruit shape QTLs.     

Identification and molecular genetic mapping of <Emphasis Type="Italic">Chili veinal mottle virus</Emphasis> (ChiVMV) resistance genes in pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>)

Chili veinal mottle virus (ChiVMV) threatens the agricultural production of peppers (Capsicum annuum) in Asia and Africa. In this study, we evaluated ChiVMV resistance in the four pepper varieties CV3, CV4, CV8, and CV9. Segregation analyses revealed that CV3 and CV8 contain the single dominant resistance gene Cvr1, and CV9 contains the single recessive resistance gene cvr4. SNP markers were developed and used to map the Cvr1 gene in CV3 to the short arm of chromosome 6 where NLR genes are clustered. In CV4 oligogenic resistance loci were detected. A genotyping-by-sequencing (GBS) combined with modified sliding window approach mapped two resistance loci, to chromosomes 6 and 10. The development of SNP markers and the resulting knowledge of genomic positioning will assist in breeding ChiVMV-resistant pepper varieties and in the fine mapping of ChiVMV resistance genes.     

Induced androgenesis of <Emphasis Type="Italic">Capsicum frutescens</Emphasis> L.

The aim of the research was to make a preliminary determination of the effectiveness of the induction of haploids in Capsicum frutescens L. In order to induce androgenesis red and yellow fruit forms of species were used, each bred by the researchers on their own. The experiment was performed in October. Anther cultures were conducted according to a modified method developed by Dumas et al. (1981) for C. annuum L. The anthers were laid on CP medium containing 0.01 mg dm⁻³ 2.4-D and 0.01 mg dm⁻³ kinetin, with the addition of 0.5 g dm⁻³ of activated carbon and 5 mg×dm⁻³ of silver nitrate, solidified with 8 g dm⁻³ of agar. The cultures were incubated in the dark at 35 deg C for 8 days. Next they were transferred to 25 deg C under a 12-hour photoperiod. After 14 days of induction, anthers were transferred to R₁ medium supplemented with 0.1 mg dm⁻³ kinetin. Obtained embryos were subsequently transplanted onto V₃ hormone-free medium and well growing plants were planted in greenhouses. The efficiency of androgenesis for both C. frutescens L. forms was relatively low and it did not exceed 5%. The ploidy level of the resulting plants was determined by flow-cytometric analysis. The regenerants consisted of about equal numbers of haploids and diploids. Additionally, among plants regenerated from anthers of yellow fruit forms, two mixoploids were observed.     

Transgenic resistance to <Emphasis Type="Italic">Cymbidium mosaic virus</Emphasis> in <Emphasis Type="Italic">Dendrobium</Emphasis> expressing the viral capsid protein gene

A Taiwan isolate of Cymbidium mosaic virus (CymMV-CS) was isolated from infected Cymbidium sinesis Willd. The cDNA of the capsid protein (CP) gene was synthesized and sequenced. Alignment of this CP gene with other reported CPs revealed homologies of 92–98% at the nucleotide level and 98–99% at the amino acid level. To generate virus-resistant varieties, the CymMV-CS CP gene was transformed into Dendrobium protocorms through particle bombardment. Transformants were selected on medium supplemented with 20 mg/L hygromycin and the presence of the transgene was confirmed by polymerase chain reaction, Southern, Northern and Western blot analyses. Transgenic Dendrobium harboring the CymMV CP gene expressed a very low level of virus accumulation four months post-inoculation with CymMV, as detected by ELISA. The transgenic plants exhibited much milder symptoms than the non-transgenic plants upon challenge with CymMV virionsSequence data reported from this article have been deposited at the GenBank Data Libraries under Accession No. AY429021.     

Genetic mapping of the novel <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> resistance gene <Emphasis Type="Italic">TuRB03</Emphasis> in <Emphasis Type="Italic">Brassica napus</Emphasis>

A new source of resistance to the pathotype 4 isolate of Turnip mosaic virus (TuMV) CDN 1 has been identified in Brassica napus (oilseed rape). Analysis of segregation of resistance to TuMV isolate CDN 1 in a backcross generation following a cross between a resistant and a susceptible B. napus line showed that the resistance was dominant and monogenic. Molecular markers linked to this dominant resistance were identified using amplified fragment length polymorphism (AFLP) and microsatellite bulk segregant analysis. Bulks consisted of individuals from a BC₁ population with the resistant or the susceptible phenotype following challenge with CDN 1. One AFLP and six microsatellite markers were associated with the resistance locus, named TuRB03, and these mapped to the same region on chromosome N6 as a previously mapped TuMV resistance gene TuRB01. Further testing of TuRB03 with other TuMV isolates showed that it was not effective against all pathotype 4 isolates. It was effective against some, but not all pathotype 3 isolates tested. It provided further resolution of TuMV pathotypes by sub-dividing pathotypes 3 and 4. TuRB03 also provides a new source of resistance for combining with other resistances in our attempts to generate durable resistance to this virus.     

Expression of an extracellular ribonuclease gene increases resistance to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> in tobacco

Background

The apoplast plays an important role in plant defense against pathogens. Some extracellular PR-4 proteins possess ribonuclease activity and may directly inhibit the growth of pathogenic fungi. It is likely that extracellular RNases can also protect plants against some viruses with RNA genomes. However, many plant RNases are multifunctional and the direct link between their ribonucleolytic activity and antiviral defense still needs to be clarified. In this study, we evaluated the resistance of Nicotiana tabacum plants expressing a non-plant single-strand-specific extracellular RNase against Cucumber mosaic virus.

Results

Severe mosaic symptoms and shrinkage were observed in the control non-transgenic plants 10 days after inoculation with Cucumber mosaic virus (CMV), whereas such disease symptoms were suppressed in the transgenic plants expressing the RNase gene. In a Western blot analysis, viral proliferation was observed in the uninoculated upper leaves of control plants, whereas virus levels were very low in those of transgenic plants. These results suggest that resistance against CMV was increased by the expression of the heterologous RNase gene.

Conclusion

We have previously shown that tobacco plants expressing heterologous RNases are characterized by high resistance to Tobacco mosaic virus. In this study, we demonstrated that elevated levels of extracellular RNase activity resulted in increased resistance to a virus with a different genome organization and life cycle. Thus, we conclude that the pathogen-induced expression of plant apoplastic RNases may increase non-specific resistance against viruses with RNA genomes.

     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l⁻¹ phosphinothricin (PPT) and 150 mg l⁻¹ kanamycin, respectively, for shoot induction and 1 mg l⁻¹ PPT and 125 mg l⁻¹ kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T₀ and T₁ plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T₀ and T₁ transgenic plants.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.     

Selection of atrazine-resistant plants by <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis in pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>)

The effects of atrazine on cotyledon cultures of Capsicum annuum (L.) cv. G₄ were investigated with a view of establishing a system for in vitro selection of resistant mutants. At low levels of herbicide produced little growth inhibition, some chlorophyll loss occurred associated with the production of albino shoots. At 20 mg l⁻¹ atrazine bleaching was more pronounced and was accompanied by the development of necrotic spots; however, efficient bleaching was associated with severe suppression of growth. Mutagenized cotyledon explants resulted in production of herbicide-resistant plants on medium containing selective levels of sucrose (0.5%) and atrazine (20 mg l⁻¹). Differential morphogenetic responses were observed when the levels of sucrose (0.5–5%) were altered. Shoot regeneration was maximum in 2 sucrose and the regenerating ability decreased with a further increase in sucrose concentration (3%–5%). However, lowering of sucrose concentration from 2 to 0.5% caused complete bleaching of explants and permitted the selection of herbicide-resistant plants. Complete atrazine-resistant plantlets were obtained after rooting of regenerated green shoots on rooting medium containing 10 mg l⁻¹atrazine, 1.0 mg l⁻¹IAA and 0.5% sucrose. Leaf-segment assay of differentiated plants revealed that all regenerants were resistant to the atrazine. Reciprocal crosses between atrazine-resistant and -sensitive plants showed a non-Mendelian transmission of resistance trait.     

Adaptation of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> soybean strains (SSVs) to cultivated and wild soybeans

Cucumber mosaic virus soybean strains formerly called soybean stunt virus (SSV) were inoculated onto 23 wild soybeans collected from four Asian countries to investigate their infectivity in order to improve understanding of the co-evolution of SSVs and soybean. SSV inoculation resulted in systemic infection in most of the wild soybeans used. However, an SSV strain (SSV-In), which was isolated in Indonesia, did not result in systemic infection of many of the wild soybeans distributed in southern Japan. This exceptional infectivity of SSV-In may be due to its specific adaptation to the local soybean population(s) of Indonesia, which has rarely been affected by gene flows from wild soybean. In the present study, the nucleotide sequences of the 3a and CP genes of SSV were determined, and the data were used to classify seven SSV isolates among known Cucumber mosaic virus (CMV) strains. The phylogenetic analysis showed that the seven SSVs formed a distinct cluster separated from the other CMV strains despite their different geographical origins; SSV-In was the most divergent of the seven isolates. Comparison of the rates of synonymous and nonsynonymous substitutions revealed that the SSV group had evolved faster than subgroup IA. The implications of the findings are discussed in relation to the so-called Red Queen hypothesis.     

A lectin with antifungal and mitogenic activities from red cluster pepper (<Emphasis Type="Italic">Capsicum frutescens</Emphasis>) seeds

A monomeric mannose/glucose-binding lectin, with a molecular mass of 29.5 kDa and an N-terminal sequence GQRELKL showing resemblance to that of the lectin-like oxidized low-density lipoprotein receptor from the rabbit, has been isolated from the seeds of red cluster pepper Capsium frutescens L. var. fasciculatum. The protocol involved anion exchange chromatography on diethylamino ethanol-cellulose and Q-Sepharose and fast protein liquid chromatography on Mono Q. Its hemagglutinating activity toward rabbit erythrocytes was inhibited by d-mannose and glucose, specifically. The activity was stable from 0 to 40°C, reached a maximum at pH 7 and 8, and was potentiated by Ca²⁺ and Mn²⁺ ions. The lectin showed strong mitogenic activity toward spleen cells isolated from BALB/c mice. The mitogenic activity, which reached a peak at a lectin concentration of 0.27 μM, was inhibited specifically by d(+)-mannose. The lectin was capable of inhibiting the germination of Aspergillus flavus and Fusarium moniliforme spores and hyphal growth in the two fungi.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying -glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies.     

Changes in leaf tissue of <Emphasis Type="Italic">Carica papaya</Emphasis> during single and mixed infections with <Emphasis Type="Italic">Papaya ringspot virus</Emphasis> and <Emphasis Type="Italic">Papaya mosaic virus</Emphasis>

Papaya (Carica papaya L.) is susceptible to viral diseases caused by Papaya mosaic virus (PapMV) and Papaya ringspot virus (PRSV), which limit fruit production and affect economic yield. The symptoms produced by both the viruses are similar in early stages of infection and include vein and leaf chlorosis, which develop into mosaic at later stages of infection when leaf lamina can get reduced in size and distorted with a shoe-string aspect. Digital image analyses, such as fractal dimension (FD) and lacunarity (λ) were used here to examine papaya tissue after single and mixed infections of PRSV and PapMV. Morphological changes, such as hypoplasia, hyperplasia, and neoplasia are described in tissues with viral infections. Furthermore, we quantified these changes and suggest three ranges of tissue damage according to their λ values in rank 1 (0.01 to 0.39), rank 2 (0.4 to 0.79), and rank 3 (0.8 to 1). Our analyses suggest that in synergism and antagonism there is a competition of both viruses to occupy the same mesophyll cells, as indicated by their intermediate values of lacunarity.     

RNAi-mediated <Emphasis Type="Italic">Soybean mosaic virus</Emphasis> (SMV) resistance of a Korean Soybean cultivar

Soybean [Glycine max (L.) Merr.] is an important crop for vegetable oil production, and is a major protein source worldwide. Because of its importance as a crop, genetic transformation has been used extensively to improve its valuable traits. Soybean mosaic virus (SMV) is one of the most well-known viral diseases affecting soybean. Transgenic soybean plants with improved resistance to SMV were produced by introducing HC-Pro coding sequences within RNA interference (RNAi) inducing hairpin construct via Agrobacterium-mediated transformation. During an experiment to confirm the response of transgenic plants (T₂) to SMV infection, no T₂ plants from lines #2 (31/31), #5 (35/35) or #6 (37/37) exhibited any SMV symptoms, indicating strong viral resistance (R), whereas NT (non-transgenic wild type) plants and those from lines #1, #3 and #4 exhibited mild mosaic (mM) or mosaic (M) symptoms. The northern blot analysis showed that three resistant lines (#2, #5 and #6) did not show the detection of viral RNA accumulation while NT, EV (transformed with empty vector carrying only Bar) and lines #1, #3 and #4 plants were detected. T₃ seeds from SMV-inoculated T₂ plants were harvested and checked for changes in seed morphology due to viral infection. T₃ seeds of lines #2, #5 and #6 were clear and seed coat mottling was not present, which is indicative of SMV resistance. RT-PCR and quantitative real-time PCR showed that T₃ seeds from the SMV-resistant lines #2, #5 and #6 did not exhibit any detection of viral RNA accumulation (HC-Pro, CP and CI), while the viral RNA accumulation was detected in SMV-susceptible lines #1, #3 and #4 plants. During the greenhouse test for viral resistance and yield components, T₃ plants from the SMV-inoculated transgenic lines #2, #5 and #6 showed viral resistance (R) and exhibited a more favorable average plant height, number of nodes per plant, number of branches per plant, number of pods per plant and total seed weight with statistical significance during strong artificial SMV infection than did other plant lines. In particular, the SMV-resistant line #2 exhibited superior average plant height, pod number and total seed weight with highly significance. According to our results, RNAi induced by the hairpin construct of the SMV HC-Pro sequence effectively confers much stronger viral resistance than did the methods used during previous trials, and has the potential to increase yields significantly. Because of its efficiency, the induction of RNAi-mediated resistance will likely be used more frequently as part of the genetic engineering of plants for crop improvement.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of poinsettia, <Emphasis Type="Italic">Euphorbia pulcherrima</Emphasis>, with virus-derived hairpin RNA constructs confers resistance to <Emphasis Type="Italic">Poinsettia mosaic virus</Emphasis>

Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2-3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.