Publications by authors named "Yuling Bai"

61 Publications

Identification of Novel Loci and Candidate Genes for Cucumber Downy Mildew Resistance Using GWAS.

Plants (Basel) 2020 Nov 27;9(12). Epub 2020 Nov 27.

Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.

Downy mildew (DM) is one of the most serious diseases in cucumber. Multiple quantitative trait loci (QTLs) for DM resistance have been detected in a limited number of cucumber accessions. In this study we applied genome-wide association analysis (GWAS) to detected genetic loci for DM resistance in a core germplasm (CG) of cucumber lines that represent diverse origins and ecotypes. Phenotypic data on responses to DM infection were collected in four field trials across three years, 2014, 2015, and 2016. With the resequencing data of these CG lines, GWAS for DM resistance was performed and detected 18 loci that were distributed on all the seven cucumber chromosomes. Of these 18 loci, only six (, and ) were detected in two experiments, and were considered as loci with a stable effect on DM resistance. Further, 16 out of the 18 loci colocalized with the QTLs that were reported in previous studies and two loci, and , were novel ones identified only in this study. Based on the annotation of homologous genes in and pairwise LD correlation analysis, several candidate genes were identified as potential causal genes underlying the stable and novel loci, including for , for , for , for , and for . This study shows that the CG germplasm is a very valuable resource carrying known and novel QTLs for DM resistance. The potential of using these CG lines for future allele-mining of candidate genes was discussed in the context of breeding cucumber with resistance to DM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/plants9121659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768435PMC
November 2020

Analysis of QTL DM4.1 for Downy Mildew Resistance in Cucumber Reveals Multiple subQTL: A Novel as Candidate Gene for the Most Important subQTL.

Front Plant Sci 2020 22;11:569876. Epub 2020 Oct 22.

Plant Breeding, Wageningen University & Research, Wageningen, Netherlands.

One of the biggest problems in cucumber cultivation is cucurbit downy mildew (DM), caused by the obligate biotroph . Whereas DM in cucumber was previously efficiently controlled by the gene from Indian cucumber accession PI 197087, this resistance was broken by new DM strains, prompting the search for novel sources of resistance. A promising source of resistance is the wild cucumber accession PI 197088. It was previously shown that DM resistance in this genotype inherits polygenically. In this paper, we put the focus on one of the QTL, DM4.1 that is located on chromosome 4. QTL DM4.1 was shown to consist of three subQTL: DM4.1.1 affected pathogen-induced necrosis, DM4.1.2 was shown to have an additive effect on sporulation, and DM4.1.3 had a recessive effect on chlorosis as well as an effect on sporulation. Near-isogenic lines (NILs) were produced by introgressing the subQTLs into a susceptible cucumber line (HS279) with good horticultural traits. Transcriptomic analysis revealed that many genes in general, and defense pathway genes in particular, were differentially expressed in NIL DM4.1.1/.2 compared to NIL DM4.1.3 and the susceptible parent HS279. This indicates that the resistance from subQTL DM4.1.1 and/or subQTL DM4.1.2 likely involves defense signaling pathways, whereas resistance due to subQTL DM4.1.3 is more likely to be independent of known defense pathways. Based on fine-mapping data, we identified the gene as a likely candidate for subQTL DM4.1.2, as this gene was found to have a loss-of-function mutation in the susceptible parent HS279, and was strongly upregulated by inoculation in NIL DM4.1.1/.2. Heterologous expression of this gene triggered necrosis, providing further evidence that this gene is indeed causal for subQTL DM4.1.2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2020.569876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7649820PMC
October 2020

Comparative genomics reveals the in planta-secreted Verticillium dahliae Av2 effector protein recognized in tomato plants that carry the V2 resistance locus.

Environ Microbiol 2020 Oct 19. Epub 2020 Oct 19.

Laboratory of Phytopathology, Wageningen University and Research, Wageningen, 6708 PB, The Netherlands.

Plant pathogens secrete effector molecules during host invasion to promote colonization. However, some of these effectors become recognized by host receptors to mount a defence response and establish immunity. Recently, a novel resistance was identified in wild tomato, mediated by the single dominant V2 locus, to control strains of the soil-borne vascular wilt fungus Verticillium dahliae that belong to race 2. With comparative genomics of race 2 strains and resistance-breaking race 3 strains, we identified the avirulence effector that activates V2 resistance, termed Av2. We identified 277 kb of race 2-specific sequence comprising only two genes encoding predicted secreted proteins that are expressed during tomato colonization. Subsequent functional analysis based on genetic complementation into race 3 isolates and targeted deletion from the race 1 isolate JR2 and race 2 isolate TO22 confirmed that one of the two candidates encodes the avirulence effector Av2 that is recognized in V2 tomato plants. Two Av2 allelic variants were identified that encode Av2 variants that differ by a single acid. Thus far, a role in virulence could not be demonstrated for either of the two variants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1462-2920.15288DOI Listing
October 2020

The NLR Protein Encoded by the Resistance Gene Is Triggered by the Replication-Associated Protein Rep/C1 of Tomato Yellow Leaf Curl Virus.

Front Plant Sci 2020 10;11:545306. Epub 2020 Sep 10.

Plant Breeding, Wageningen University & Research, Wageningen, Netherlands.

The whitefly-transmitted tomato yellow leaf curl virus (TYLCV) is one of the most destructive viral pathogens of cultivated tomato. To combat TYLCV, resistance gene has been introduced into cultivated tomato () from wild tomato species by interspecific crossing. Introgression lines with contain a large inversion compared with , which causes severe suppression of recombination and has hampered the cloning of so far. Here, we report the fine-mapping and cloning of using crosses between a introgression line and several susceptible accessions. was shown to encode a nucleotide-binding leucine-rich repeat (NLR) protein. For breeding purposes, a highly specific DNA marker tightly linked to the gene was developed permitting marker-assisted selection. The resistance mediated by was effective against the Israel strain of TYLCV (TYLCV-IL) and tomato yellow leaf curl virus-[China : Shanghai2] (TYLCV-[CN : SH2]), but not against tomato yellow leaf curl Sardinia virus (TYLCSV) and leafhopper-transmitted beet curly top virus (BCTV). By co-infiltration experiments we showed that transient expression of the Rep/C1 protein of TYLCV, but not of TYLCSV triggered a hypersensitive response (HR) in plants co-expressing the gene. Our results indicate that the / gene of TYLCV-IL presents the avirulence determinant of mediated resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2020.545306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511541PMC
September 2020

CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew.

BMC Plant Biol 2020 Jun 19;20(1):284. Epub 2020 Jun 19.

Plant Breeding, Wageningen University & Research, Droevendaalsesteeg 1, 6708, PB, Wageningen, The Netherlands.

Background: The development of CRISPR/Cas9 technology has facilitated targeted mutagenesis in an efficient and precise way. Previously, RNAi silencing of the susceptibility (S) gene PowderyMildewResistance 4 (PMR4) in tomato has been shown to enhance resistance against the powdery mildew pathogen Oidium neolycopersici (On).

Results: To study whether full knock-out of the tomato PMR4 gene would result in a higher level of resistance than in the RNAi-silenced transgenic plants we generated tomato PMR4 CRISPR mutants. We used a CRISPR/Cas9 construct containing four single-guide RNAs (sgRNAs) targeting the tomato PMR4 gene to increase the possibility of large deletions in the mutants. After PCR-based selection and sequencing of transformants, we identified five different mutation events, including deletions from 4 to 900-bp, a 1-bp insertion and a 892-bp inversion. These mutants all showed reduced susceptibility to On based on visual scoring of disease symptoms and quantification of relative fungal biomass. Histological observations revealed a significantly higher occurrence of hypersensitive response-like cell death at sites of fungal infection in the pmr4 mutants compared to wild-type plants. Both haustorial formation and hyphal growth were diminished but not completely inhibited in the mutants.

Conclusion: CRISPR/Cas-9 targeted mutagenesis of the tomato PMR4 gene resulted in mutants with reduced but not complete loss of susceptibility to the PM pathogen On. Our study demonstrates the efficiency and versatility of the CRISPR/Cas9 system as a powerful tool to study and characterize S-genes by generating different types of mutations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12870-020-02497-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7304142PMC
June 2020

Breeding Has Increased the Diversity of Cultivated Tomato in The Netherlands.

Front Plant Sci 2019 20;10:1606. Epub 2019 Dec 20.

Plant Breeding, Wageningen University & Research, Wageningen, Netherlands.

It is generally believed that domestication and breeding of plants has led to genetic erosion, including loss of nutritional value and resistances to diseases, especially in tomato. We studied the diversity dynamics of greenhouse tomato varieties in NW Europe, especially The Netherlands, over the last seven decades. According to the used SNP array, the genetic diversity was indeed very low during the 1960s, but is now eight times higher when compared to that dip. The pressure since the 1970s to apply less pesticides led to the introgression of many disease resistances from wild relatives, representing the first boost of genetic diversity. In Europe a second boost ensued, largely driven by German popular media who named poor tasting tomatoes (water bombs). The subsequent collapse of Dutch tomato exports to Germany fueled breeding for fruit flavor, further increasing diversity since the 1990s. The increased diversity in composition of aroma volatiles observed starting from 1990s may reflect the efforts of breeders to improve fruit quality. Specific groups of aroma compounds showed different quantitative trend over the decades studied. Our study provides compelling evidence that breeding has increased the diversity of tomato varieties considerably since the 1970s.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2019.01606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6932954PMC
December 2019

Ty-1, a universal resistance gene against geminiviruses that is compromised by co-replication of a betasatellite.

Mol Plant Pathol 2020 02 22;21(2):160-172. Epub 2019 Nov 22.

Laboratory of Virology, Wageningen University & Research, Droevendaalsesteeg 1, Wageningen, 6708PB, Netherlands.

Tomato yellow leaf curl virus (TYLCV), a begomovirus, causes large yield losses and breeding for resistance is an effective way to combat this viral disease. The resistance gene Ty-1 codes for an RNA-dependent RNA polymerase and has recently been shown to enhance transcriptional gene silencing of TYLCV. Whereas Ty-1 was earlier shown to also confer resistance to a bipartite begomovirus, here it is shown that Ty-1 is probably generic to all geminiviruses. A tomato Ty-1 introgression line, but also stable transformants of susceptible tomato cv. Moneymaker and Nicotiana benthamiana (N. benthamiana) expressing the Ty-1 gene, exhibited resistance to begomoviruses as well as to the distinct, leafhopper-transmitted beet curly top virus, a curtovirus. Stable Ty-1 transformants of N. benthamiana and tomato showed fewer symptoms and reduced viral titres on infection compared to wild-type plants. TYLCV infections in wild-type N. benthamiana plants in the additional presence of a betasatellite led to increased symptom severity and a consistent, slightly lowered virus titre relative to the high averaged levels seen in the absence of the betasatellite. On the contrary, in Ty-1 transformed N. benthamiana viral titres increased in the presence of the betasatellite. The same was observed when these Ty-1-encoding plants were challenged with TYLCV and a potato virus X construct expressing the RNA interference suppressor protein βC1 encoded by the betasatellite. The resistance spectrum of Ty-1 and the durability of the resistance are discussed in light of antiviral RNA interference and viral counter defence strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/mpp.12885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988424PMC
February 2020

CRISPR/Cas inactivation of RECQ4 increases homeologous crossovers in an interspecific tomato hybrid.

Plant Biotechnol J 2020 03 30;18(3):805-813. Epub 2019 Sep 30.

Plant Breeding, Wageningen University & Research, Wageningen, The Netherlands.

Crossover formation during meiosis in plants is required for proper chromosome segregation and is essential for crop breeding as it allows an (optimal) combination of traits by mixing parental alleles on each chromosome. Crossover formation commences with the production of a large number of DNA double-strand breaks, of which only a few result in crossovers. A small number of genes, which drive the resolution of DNA crossover intermediate structures towards non-crossovers, have been identified in Arabidopisis thaliana. In order to explore the potential of modification of these genes in interspecific hybrids between crops and their wild relatives towards increased production of crossovers, we have used CRISPR/Cas9-mutagenesis in an interspecific tomato hybrid to knockout RecQ4. A biallelic recq4 mutant was obtained in the F1 hybrid of Solanum lycopersicum and S. pimpinellifolium. Compared with the wild-type F1 hybrid, the F1 recq4 mutant was shown to have a significant increase in crossovers: a 1.53-fold increase when directly observing ring bivalents in male meiocytes microscopically and a 1.8-fold extension of the genetic map when measured by analysing SNP markers in the progeny (F2) plants. This is one of the first demonstrations of increasing crossover frequency in interspecific hybrids by manipulating genes in crossover intermediate resolution pathways and the first to do so by directed mutagenesis. SIGNIFICANCE STATEMENT: Increasing crossover frequency during meiosis can speed up or simplify crop breeding that relies on meiotic crossovers to introduce favourable alleles controlling important traits from wild relatives into crops. Here we show for the first time that knocking out an inhibitor of crossovers in an interspecific hybrid between tomato and its relative wild species using CRISPR/Cas9-mutagenesis results in increased recombination between the two genomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/pbi.13248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7004904PMC
March 2020

Resistance to Tomato Yellow Leaf Curl Virus in Tomato Germplasm.

Front Plant Sci 2018 20;9:1198. Epub 2018 Aug 20.

The Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China.

Tomato yellow leaf curl virus (TYLCV) is a virus species causing epidemics in tomato () worldwide. Many efforts have been focused on identification of resistance sources by screening wild tomato species. In many cases, the accession numbers were either not provided in publications or not provided in a consistent manner, which led to redundant screenings. In the current study, we summarized efforts on the screenings of wild tomato species for TYLCV resistance from various publications. In addition, we screened 708 accessions from 13 wild tomato species using different inoculation assays (i.e., whitefly natural infection and Agrobacterium-mediated inoculation) from which 138 accessions exhibited no tomato yellow leaf curl disease (TYLCD) symptoms. These symptomless accessions include 14 accessions from , 43 from , 1 from , 28 from , 5 from , 4 from , 2 from , 1 from , 39 from , and 1 from . Most of the screened accessions remained symptomless. Many symptomless accessions were also identified in , , and . A large number of accessions were screened. However, almost all of the tested accessions showed TYLCD symptoms. Further, we studied allelic variation of the / gene in few accessions by applying virus-induced gene silencing and allele mining, leading to identification of a number of allele-specific polymorphisms. Taken together, we present a comprehensive overview on TYLCV resistance and susceptibility in wild tomato germplasm, and demonstrate how to study allelic variants of the cloned -genes in TYLCV-resistant accessions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2018.01198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110163PMC
August 2018

The Role of Tomato Genes in Plant Responses to Combined Abiotic and Biotic Stresses.

Front Plant Sci 2018 13;9:801. Epub 2018 Jun 13.

Plant Breeding, Wageningen University & Research, Wageningen, Netherlands.

In the field, plants constantly face a plethora of abiotic and biotic stresses that can impart detrimental effects on plants. In response to multiple stresses, plants can rapidly reprogram their transcriptome through a tightly regulated and highly dynamic regulatory network where WRKY transcription factors can act as activators or repressors. WRKY transcription factors have diverse biological functions in plants, but most notably are key players in plant responses to biotic and abiotic stresses. In tomato there are 83 genes identified. Here we review recent progress on functions of these tomato genes and their homologs in other plant species, such as Arabidopsis and rice, with a special focus on their involvement in responses to abiotic and biotic stresses. In particular, we highlight genes that play a role in plant responses to a combination of abiotic and biotic stresses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2018.00801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6008426PMC
June 2018

Plant behaviour under combined stress: tomato responses to combined salinity and pathogen stress.

Plant J 2018 02 19;93(4):781-793. Epub 2018 Jan 19.

Plant Breeding, Wageningen University & Research, P.O. Box 386, Wageningen, 6700AJ, The Netherlands.

Crop plants are subjected to a variety of stresses during their lifecycle, including abiotic stress factors such as salinity and biotic stress factors such as pathogens. Plants have developed a multitude of defense and adaptation responses to these stress factors. In the field, different stress factors mostly occur concurrently resulting in a new state of stress, the combined stress. There is evidence that plant resistance to pathogens can be attenuated or enhanced by abiotic stress factors. With stress tolerance research being mostly focused on plant responses to individual stresses, the understanding of a plant's ability to adapt to combined stresses is limited. In the last few years, we studied powdery mildew resistance under salt stress conditions in the model crop plant tomato with the aim to understand the requirements to achieve plant resilience to a wider array of combined abiotic and biotic stress combinations. We uncovered specific responses of tomato plants to combined salinity-pathogen stress, which varied with salinity intensity and plant resistance genes. Moreover, hormones, with their complex regulation and cross-talk, were shown to play a key role in the adaptation of tomato plants to the combined stress. In this review, we attempt to understand the complexity of plant responses to abiotic and biotic stress combinations, with a focus on tomato responses (genetic control and cross-talk of signaling pathways) to combined salinity and pathogen stresses. Further, we provide recommendations on how to design novel strategies for breeding crops with a sustained performance under diverse environmental conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/tpj.13800DOI Listing
February 2018

Silencing of DND1 in potato and tomato impedes conidial germination, attachment and hyphal growth of Botrytis cinerea.

BMC Plant Biol 2017 Dec 6;17(1):235. Epub 2017 Dec 6.

Plant Breeding, Wageningen University & Research, Droevendaalsesteeg 1, 6708, PB, Wageningen, The Netherlands.

Background: Botrytis cinerea, a necrotrophic pathogenic fungus, attacks many crops including potato and tomato. Major genes for complete resistance to B. cinerea are not known in plants, but a few quantitative trait loci have been described in tomato. Loss of function of particular susceptibility (S) genes appears to provide a new source of resistance to B. cinerea in Arabidopsis.

Results: In this study, orthologs of Arabidopsis S genes (DND1, DMR6, DMR1 and PMR4) were silenced by RNAi in potato and tomato (only for DND1). DND1 well-silenced potato and tomato plants showed significantly reduced diameters of B. cinerea lesions as compared to control plants, at all-time points analysed. Reduced lesion diameter was also observed on leaves of DMR6 silenced potato plants but only at 3 days post inoculation (dpi). The DMR1 and PMR4 silenced potato transformants were as susceptible as the control cv Desiree. Microscopic analysis was performed to observe B. cinerea infection progress in DND1 well-silenced potato and tomato leaves. A significantly lower number of B. cinerea conidia remained attached to the leaf surface of DND1 well-silenced potato and tomato plants and the hyphal growth of germlings was hampered.

Conclusions: This is the first report of a cytological investigation of Botrytis development on DND1-silenced crop plants. Silencing of DND1 led to reduced susceptibility to Botrytis, which was associated with impediment of conidial germination and attachment as well as hyphal growth. Our results provide new insights regarding the use of S genes in resistance breeding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12870-017-1184-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719932PMC
December 2017

Functional Characterization of a Syntaxin Involved in Tomato () Resistance against Powdery Mildew.

Front Plant Sci 2017 20;8:1573. Epub 2017 Sep 20.

Plant Breeding, Wageningen University & ResearchWageningen, Netherlands.

Specific syntaxins, such as Arabidopsis AtPEN1 and its barley ortholog ROR2, play a major role in plant defense against powdery mildews. Indeed, the impairment of these genes results in increased fungal penetration in both host and non-host interactions. In this study, a genome-wide survey allowed the identification of 21 tomato syntaxins. Two of them, named and , are closely related to . RNAi-based silencing of in a tomato line carrying a loss-of-function mutation of the susceptibility gene led to compromised resistance toward the tomato powdery mildew fungus . Moreover, it resulted in a significant increase in the penetration rate of the non-adapted powdery mildew fungus f. sp. . Codon-based evolutionary analysis and multiple alignments allowed the detection of amino acid residues that are under purifying selection and are specifically conserved in syntaxins involved in plant-powdery mildew interactions. Our findings provide both insights on the evolution of syntaxins and information about their function which is of interest for future studies on plant-pathogen interactions and tomato breeding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2017.01573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5611543PMC
September 2017

Key Components of Different Plant Defense Pathways Are Dispensable for Powdery Mildew Resistance of the Arabidopsis Triple Mutant.

Front Plant Sci 2017 19;8:1006. Epub 2017 Jun 19.

Unit of Plant Molecular Cell Biology, Institute for Biology I, RWTH Aachen UniversityAachen, Germany.

Loss of function mutations of particular plant () genes confer durable and broad-spectrum penetration resistance against powdery mildew fungi. Here, we combined genetic, transcriptomic and metabolomic analyses to explore the defense mechanisms in the fully resistant triple mutant. We found that this genotype unexpectedly overcomes the requirement for indolic antimicrobials and defense-related secretion, which are critical for incomplete resistance of single mutants. Comparative microarray-based transcriptome analysis of mutants and wild type plants upon inoculation revealed an increased and accelerated accumulation of many defense-related transcripts. Despite the biotrophic nature of the interaction, this included the non-canonical activation of a jasmonic acid/ethylene-dependent transcriptional program. In contrast to a non-adapted powdery mildew pathogen, the adapted powdery mildew fungus is able to defeat the accumulation of defense-relevant indolic metabolites in a MLO protein-dependent manner. We suggest that a broad and fast activation of immune responses in plants can compensate for the lack of single or few defense pathways. In addition, our results point to a role of Arabidopsis MLO2, MLO6, and MLO12 in enabling defense suppression during invasion by adapted powdery mildew fungi.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2017.01006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5475338PMC
June 2017

Functional characterization of cucumber (Cucumis sativus L.) Clade V MLO genes.

BMC Plant Biol 2017 04 21;17(1):80. Epub 2017 Apr 21.

Plant Breeding, Wageningen University & Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.

Background: Powdery mildew (PM) causing fungi are well-known pathogens, infecting over 10.000 plant species, including the economically important crop cucumber (Cucumis sativus L.). Loss-of-function mutations in clade V MLO genes have previously been shown to lead to recessively inherited broad-spectrum resistance to PM in several species. In cucumber, one clade V MLO homolog (CsaMLO8) was previously identified as being a susceptibility factor to PM. Two other closely related homologs (CsaMLO1 and CsaMLO11) were found, but their function was not yet unravelled.

Methods: CsaMLO1 and CsaMLO11 were cloned from cucumber and overexpressed in a tomato mlo mutant. The transcript abundances of all three CsaMLO genes in different cucumber tissues were quantified using qRT-PCR and RNA-seq, with and without inoculation with the cucumber PM fungus Podosphaera xanthii. Allelic variation of CsaMLO1 and CsaMLO11 was screened in silico in sequenced cucumber germplasm.

Results: Heterologous overexpression of all three CsaMLO genes in the tomato mlo mutant restored susceptibility to PM caused by Oidium neolycopersici, albeit to a different extent: whereas overexpression of CsaMLO1 or CsaMLO8 completely restored susceptibility, overexpression of CsaMLO11 was only partially able to restore PM susceptibility. Furthermore, it was observed by qRT-PCR and RNA-seq that CsaMLO8 was significantly higher expressed in non-inoculated cucumber compared to the other two MLO genes. However, inoculation with P. xanthii led to upregulation of CsaMLO1, but not to upregulation of CsaMLO8 or CsaMLO11.

Conclusions: Both CsaMLO1 and CsaMLO11 are functional susceptibility genes, although we conclude that based on the transcript abundance CsaMLO8 is probably the major clade V MLO gene in cucumber regarding providing susceptibility to PM. Potential loss-of-function mutations in CsaMLO1 and CsaMLO11 have not been identified. The generation and analysis of such mutants are interesting subjects for further investigation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12870-017-1029-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5399834PMC
April 2017

Frequency of a natural truncated allele of in the germplasm of .

Mol Breed 2017 4;37(1). Epub 2017 Jan 4.

Research and Innovation Centre, Fondazione Edmund Mach, Via Edmund Mach 1, 38010 San Michele all'Adige, Italy.

is the causal agent of powdery mildew (PM) in apple. To reduce the amount of fungicides required to control this pathogen, the development of resistant apple cultivars should become a priority. Resistance to PM was achieved in various crops by knocking out specific members of the gene family that are responsible for PM susceptibility (S-genes). In apple, the knockdown of resulted in PM resistance. However, since gene silencing technologies such as RNAi are perceived unfavorably in Europe, a different approach that exploits this type of resistance is needed. This work evaluates the presence of non-functional naturally occurring alleles of in apple germplasm. The screening of the re-sequencing data of 63 apple individuals led to the identification of 627 single nucleotide polymorphisms (SNPs) in five genes (, and ), 127 of which were located in exons. The T-1201 insertion of a single nucleotide in caused the formation of an early stop codon, resulting in a truncated protein lacking 185 amino acids, including the calmodulin-binding domain. The presence of the insertion was evaluated in 115 individuals. It was heterozygous in 64 and homozygous in 25. Twelve of the 25 individuals carrying the insertion in homozygosity were susceptible to PM. After barley, pea, cucumber, and tomato, apple would be the fifth species for which a natural non-functional allele has been found.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11032-016-0610-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5214879PMC
January 2017

Ethylene and Abscisic Acid Signaling Pathways Differentially Influence Tomato Resistance to Combined Powdery Mildew and Salt Stress.

Front Plant Sci 2016 9;7:2009. Epub 2017 Jan 9.

Plant Breeding, Wageningen University & Research Wageningen, Netherlands.

There is currently limited knowledge on the role of hormones in plants responses to combinations of abiotic and pathogen stress factors. This study focused on the response of tomato near-isogenic lines (NILs) that carry the , and loci, conferring resistance to tomato powdery mildew (PM) caused by , to combined PM and salt stress. These NILs were crossed with the (ABA-deficient), (JA-deficient), and (ET overproducer) tomato mutants to investigate possible roles of hormone signaling in response to combined stresses. In the NILs, marker genes for hormonal pathways showed differential expression patterns upon PM infection. The mutation resulted in breakdown of resistance in NIL-Ol-1 and NIL-ol-2. This was accompanied by reduced callose deposition, and was more pronounced under combined salt stress. The mutation resulted in HO overproduction and reduced susceptibility to PM in NIL-Ol-1 under combined stress, but lead to higher plant growth reduction under salinity and combined stress. Resistance in NIL-ol-2 was compromised by the mutation, which was potentially caused by reduction of callose deposition. Under combined stress the compromised resistance in NIL-ol-2 was restored. PM resistance in NIL-Ol-4 remained robust across all mutant and treatment combinations. Hormone signaling is critical to the response to combined stress and PM, in terms of resistance and plant fitness. ABA appears to be at the crossroads of disease susceptibility/senescence and plant performance under combined stress These gained insights can aid in narrowing down targets for improving crop performance under stress combinations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2016.02009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5220069PMC
January 2017

Functional characterization of the powdery mildew susceptibility gene SmMLO1 in eggplant (Solanum melongena L.).

Transgenic Res 2017 06 9;26(3):323-330. Epub 2017 Jan 9.

Section of Genetics and Plant Breeding, Department of Plant, Soil and Food Science, University of Bari "Aldo Moro", Via Amendola 165/A, 70126, Bari, Italy.

Eggplant (Solanum melongena L.) is one of the most important vegetables among the Solanaceae and can be a host to fungal species causing powdery mildew (PM) disease. Specific homologs of the plant Mildew Locus O (MLO) gene family are PM susceptibility factors, as their loss of function results in a recessive form of resistance known as mlo resistance. In a previous work, we isolated the eggplant MLO homolog SmMLO1. SmMLO1 is closely related to MLO susceptibility genes characterized in other plant species. However, it displays a peculiar non-synonymous substitution that leads to a T → M amino acid change at protein position 422, in correspondence of the MLO calmodulin-binding domain. In this study, we performed the functional characterization of SmMLO1. Transgenic overexpression of SmMLO1 in a tomato mlo mutant compromised resistance to the tomato PM pathogen Oidium neolycopersici, thus indicating that SmMLO1 is a PM susceptibility factor in eggplant. PM susceptibility was also restored by the transgenic expression of a synthetic gene, named s-SmMLO1, encoding a protein identical to SmMLO1, except for the presence of T at position 422. This indicates that the T → M polymorphism does not affect the protein role as PM susceptibility factor. Overall, the results of this work are of interest for the functional characterization of MLO proteins and the introduction of PM resistance in eggplant using reverse genetics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11248-016-0007-9DOI Listing
June 2017

Genome-Wide Study of the Tomato SlMLO Gene Family and Its Functional Characterization in Response to the Powdery Mildew Fungus Oidium neolycopersici.

Front Plant Sci 2016 6;7:380. Epub 2016 Apr 6.

Wageningen UR Plant Breeding, Wageningen University and Research Centre Wageningen, Netherlands.

The MLO (Mildew Locus O) gene family encodes plant-specific proteins containing seven transmembrane domains and likely acting in signal transduction in a calcium and calmodulin dependent manner. Some members of the MLO family are susceptibility factors toward fungi causing the powdery mildew disease. In tomato, for example, the loss-of-function of the MLO gene SlMLO1 leads to a particular form of powdery mildew resistance, called ol-2, which arrests almost completely fungal penetration. This type of penetration resistance is characterized by the apposition of papillae at the sites of plant-pathogen interaction. Other MLO homologs in Arabidopsis regulate root response to mechanical stimuli (AtMLO4 and AtMLO11) and pollen tube reception by the female gametophyte (AtMLO7). However, the role of most MLO genes remains unknown. In this work, we provide a genome-wide study of the tomato SlMLO gene family. Besides SlMLO1, other 15 SlMLO homologs were identified and characterized with respect to their structure, genomic organization, phylogenetic relationship, and expression profile. In addition, by analysis of transgenic plants, we demonstrated that simultaneous silencing of SlMLO1 and two of its closely related homologs, SlMLO5 and SlMLO8, confer higher level of resistance than the one associated with the ol-2 mutation. The outcome of this study provides evidence for functional redundancy among tomato homolog genes involved in powdery mildew susceptibility. Moreover, we developed a series of transgenic lines silenced for individual SlMLO homologs, which lay the foundation for further investigations aimed at assigning new biological functions to the MLO gene family.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2016.00380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986958PMC
August 2016

Responses to combined abiotic and biotic stress in tomato are governed by stress intensity and resistance mechanism.

J Exp Bot 2016 09 19;67(17):5119-32. Epub 2016 Jul 19.

Wageningen UR Plant Breeding, Wageningen University & Research Centre, PO Box 386, 6700AJ, Wageningen, The Netherlands

Stress conditions in agricultural ecosystems can occur at variable intensities. Different resistance mechanisms against abiotic stress and pathogens are deployed by plants. Thus, it is important to examine plant responses to stress combinations under different scenarios. Here, we evaluated the effect of different levels of salt stress ranging from mild to severe (50, 100, and 150mM NaCl) on powdery mildew resistance and overall performance of tomato introgression lines with contrasting levels of partial resistance, as well as near-isogenic lines (NILs) carrying the resistance gene Ol-1 (associated with a slow hypersensitivity response; HR), ol-2 (an mlo mutant associated with papilla formation), and Ol-4 (an R gene associated with a fast HR). Powdery mildew resistance was affected by salt stress in a genotype- and stress intensity-dependent manner. In susceptible and partial resistant lines, increased susceptibility was observed under mild salt stress (50mM) which was accompanied by accelerated cell death-like senescence. In contrast, severe salt stress (150mM) reduced disease symptoms. Na(+) and Cl(-) accumulation in the leaves was linearly related to the decreased pathogen symptoms under severe stress. In contrast, complete resistance mediated by ol-2 and Ol-4 was unaffected under all treatment combinations, and was associated with a decreased growth penalty. Increased susceptibility and senescence under combined stress in NIL-Ol-1 was associated with the induction of ethylene and jasmonic acid pathway genes and the cell wall invertase gene LIN6. These results highlight the significance of stress severity and resistance type on the plant's performance under the combination of abiotic and biotic stress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jxb/erw285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014164PMC
September 2016

Silencing of six susceptibility genes results in potato late blight resistance.

Transgenic Res 2016 10 28;25(5):731-42. Epub 2016 May 28.

Wageningen UR Plant Breeding, Wageningen University and Research Centre, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.

Phytophthora infestans, the causal agent of late blight, is a major threat to commercial potato production worldwide. Significant costs are required for crop protection to secure yield. Many dominant genes for resistance (R-genes) to potato late blight have been identified, and some of these R-genes have been applied in potato breeding. However, the P. infestans population rapidly accumulates new virulent strains that render R-genes ineffective. Here we introduce a new class of resistance which is based on the loss-of-function of a susceptibility gene (S-gene) encoding a product exploited by pathogens during infection and colonization. Impaired S-genes primarily result in recessive resistance traits in contrast to recognition-based resistance that is governed by dominant R-genes. In Arabidopsis thaliana, many S-genes have been detected in screens of mutant populations. In the present study, we selected 11 A. thaliana S-genes and silenced orthologous genes in the potato cultivar Desiree, which is highly susceptible to late blight. The silencing of five genes resulted in complete resistance to the P. infestans isolate Pic99189, and the silencing of a sixth S-gene resulted in reduced susceptibility. The application of S-genes to potato breeding for resistance to late blight is further discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11248-016-9964-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5023794PMC
October 2016

The knock-down of the expression of MdMLO19 reduces susceptibility to powdery mildew (Podosphaera leucotricha) in apple (Malus domestica).

Plant Biotechnol J 2016 10 11;14(10):2033-44. Epub 2016 May 11.

Research and Innovation Center, Fondazione Edmund Mach, San Michele all'Adige, Italy.

Varieties resistant to powdery mildew (PM; caused by Podosphaera leucotricha) are a major component of sustainable apple production. Resistance can be achieved by knocking-out susceptibility S-genes to be singled out among members of the MLO (Mildew Locus O) gene family. Candidates are MLO S-genes of phylogenetic clade V up-regulated upon PM inoculation, such as MdMLO11 and 19 (clade V) and MdMLO18 (clade VII). We report the knock-down through RNA interference of MdMLO11 and 19, as well as the complementation of resistance with MdMLO18 in the Arabidopsis thaliana triple mlo mutant Atmlo2/6/12. The knock-down of MdMLO19 reduced PM disease severity by 75%, whereas the knock-down of MdMLO11, alone or in combination with MdMLO19, did not result in any reduction or additional reduction of susceptibility compared with MdMLO19 alone. The test in A. thaliana excluded a role for MdMLO18 in PM susceptibility. Cell wall appositions (papillae) were present in both PM-resistant and PM-susceptible plants, but were larger in resistant lines. No obvious negative phenotype was observed in plants with mlo genes knocked down. Apparently, MdMLO19 plays the pivotal role in apple PM susceptibility and its knock-down induces a very significant level of resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043462PMC
http://dx.doi.org/10.1111/pbi.12562DOI Listing
October 2016

Down-regulation of Arabidopsis DND1 orthologs in potato and tomato leads to broad-spectrum resistance to late blight and powdery mildew.

Transgenic Res 2016 Apr 17;25(2):123-38. Epub 2015 Nov 17.

Wageningen UR Plant Breeding, Wageningen University and Research Centre, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.

Multiple susceptibility genes (S), identified in Arabidopsis, have been shown to be functionally conserved in crop plants. Mutations in these S genes result in resistance to different pathogens, opening a new way to achieve plant disease resistance. The aim of this study was to investigate the role of Defense No Death 1 (DND1) in susceptibility of tomato and potato to late blight (Phytophthora infestans). In Arabidopsis, the dnd1 mutant has broad-spectrum resistance against several fungal, bacterial, and viral pathogens. However this mutation is also associated with a dwarfed phenotype. Using an RNAi approach, we silenced AtDND1 orthologs in potato and tomato. Our results showed that silencing of the DND1 ortholog in both crops resulted in resistance to the pathogenic oomycete P. infestans and to two powdery mildew species, Oidium neolycopersici and Golovinomyces orontii. The resistance to P. infestans in potato was effective to four different isolates although the level of resistance (complete or partial) was dependent on the aggressiveness of the isolate. In tomato, DND1-silenced plants showed a severe dwarf phenotype and autonecrosis, whereas DND1-silenced potato plants were not dwarfed and showed a less pronounced autonecrosis. Our results indicate that S gene function of DND1 is conserved in tomato and potato. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of DND1 orthologs, as well as additional S gene orthologs from Arabidopsis, to breed for resistance to pathogens in crop plants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11248-015-9921-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4762934PMC
April 2016

Monocot and dicot MLO powdery mildew susceptibility factors are functionally conserved in spite of the evolution of class-specific molecular features.

BMC Plant Biol 2015 Oct 26;15:257. Epub 2015 Oct 26.

Department of Soil, Plant and Food Science, Section of Genetics and Plant Breeding, University of Bari, Via Amendola 165/A, 70126, Bari, Italy.

Background: Specific members of the plant Mildew Locus O (MLO) protein family act as susceptibility factors towards powdery mildew (PM), a worldwide-spread fungal disease threatening many cultivated species. Previous studies indicated that monocot and dicot MLO susceptibility proteins are phylogenetically divergent.

Methods: A bioinformatic approach was followed to study the type of evolution of Angiosperm MLO susceptibility proteins. Transgenic complementation tests were performed for functional analysis.

Results: Our results show that monocot and dicot MLO susceptibility proteins evolved class-specific conservation patterns. Many of them appear to be the result of negative selection and thus are likely to provide an adaptive value. We also tested whether different molecular features between monocot and dicot MLO proteins are specifically required by PM fungal species to cause pathogenesis. To this aim, we transformed a tomato mutant impaired for the endogenous SlMLO1 gene, and therefore resistant to the tomato PM species Oidium neolycopersici, with heterologous MLO susceptibility genes from the monocot barley and the dicot pea. In both cases, we observed restoration of PM symptoms. Finally, through histological observations, we demonstrate that both monocot and dicot susceptibility alleles of the MLO genes predispose to penetration of a non-adapted PM fungal species in plant epidermal cells.

Conclusions: With this study, we provide insights on the evolution and function of MLO genes involved in the interaction with PM fungi. With respect to breeding research, we show that transgenic complementation assays involving phylogenetically distant plant species can be used for the characterization of novel MLO susceptibility genes. Moreover, we provide an overview of MLO protein molecular features predicted to play a major role in PM susceptibility. These represent ideal targets for future approaches of reverse genetics, addressed to the selection of loss-of-function resistant mutants in cultivated species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12870-015-0639-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4620714PMC
October 2015

Effect of the co-culture of human bone marrow mesenchymal stromal cells with human umbilical vein endothelial cells in vitro.

J Recept Signal Transduct Res 2016 19;36(3):221-4. Epub 2015 Oct 19.

a Institute of Orthopedics and Traumatology, the Third Affiliated Hospital, Guangzhou Medical University , People's Republic of China , and.

Mesenchymal stem cells (MSCs) give origin to the marrow tromal environment that supports hematopoiesis. These cells present a wide range of differentiation potentials and a complex relationship with hematopoietic stem cells (HSCs) and endothelial cells. In addition to bone marrow (BM), MSCs can be obtained from other sites in the adult or the fetus. Recent studies have shown that cocultured endothelial cells and osteoblasts are mutually promotive in bone tissues repair. In this study, we observed the effects of coculture of endothelial cells and osteoblasts at different ratios on vasculogenesis and bone formation, and we found that angiogenic effect is more effective when endothelial cells are cocultured with osteoblasts at the ratio of 4:1, and osteogenic effect is more effective at the ratio of 1:4. It is concluded that the co-culture of human bone marrow mesenchymal stromal cells with human umbilical vein endothelial cells could be a promising culture system for bone tissue engineering applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3109/10799893.2015.1075043DOI Listing
October 2016

A transposable element insertion in the susceptibility gene CsaMLO8 results in hypocotyl resistance to powdery mildew in cucumber.

BMC Plant Biol 2015 Oct 9;15:243. Epub 2015 Oct 9.

Wageningen UR Plant Breeding, Wageningen University & Research centre, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.

Background: Powdery mildew (PM) is an important disease of cucumber (Cucumis sativus L.). CsaMLO8 was previously identified as a candidate susceptibility gene for PM in cucumber, for two reasons: 1) This gene clusters phylogenetically in clade V, which has previously been shown to harbour all known MLO-like susceptibility genes for PM identified in dicot species; 2) This gene co-localizes with a QTL on chromosome 5 for hypocotyl-specific resistance to PM.

Methods: CsaMLO8 alleles from susceptible and resistant cucumber were cloned and transformed to mlo-mutant tomato. Cucumber seedlings were inoculated with Podosphaera xanthii, tissues were studied for CsaMLO8 expression at several timepoints post inoculation using qRT-PCR. The occurrence of the observed loss-of-function allele of CsaMLO8 in resequenced cucumber accessions was studied in silico.

Results: We cloned CsaMLO8 alleles from susceptible and resistant cucumber genotypes, the latter carrying the QTL for hypocotyl resistance. We found that insertion of a non-autonomous Class LTR retrotransposable element in the resistant genotype leads to aberrant splicing of CsaMLO8 mRNA. Heterologous expression of the wild-type allele of CsaMLO8 in a tomato mlo-mutant restored PM susceptibility. However, heterologous expression of the CsaMLO8 allele cloned from the resistant cucumber genotype failed to restore PM susceptibility. Furthermore we showed that inoculation of susceptible cucumber with the PM pathogen Podosphaera xanthii induced transcriptional upregulation of CsaMLO8 in hypocotyl tissue, but not in cotyledon or leaf tissue. This coincides with the observation that the QTL at the CsaMLO8-locus causes full resistance in hypocotyl tissue, but only partial resistance in cotyledons and true leafs. We studied the occurrence of the loss-of-function allele of CsaMLO8 in cucumber germplasm by an in silico approach using resequencing data of a collection of 115 cucumber accessions, and found that this allele was present in 31 out of 115 accessions.

Conclusions: CsaMLO8 was characterised as a functional susceptibility gene to PM, particularly in the hypocotyl where it was transcriptionally upregulated upon inoculation with the PM pathogen P. xanthii. A loss-of-function mutation in CsaMLO8 due to the insertion of a transposable element was found to be the cause of hypocotyl resistance to PM. This particular allele of CsaMLO8 was found to occur in 27 % of the resequenced cucumber accessions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12870-015-0635-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4600303PMC
October 2015

Detection of an inversion in the Ty-2 region between S. lycopersicum and S. habrochaites by a combination of de novo genome assembly and BAC cloning.

Theor Appl Genet 2015 Oct 8;128(10):1987-97. Epub 2015 Jul 8.

Wageningen UR Plant Breeding, Wageningen University & Research Centre, P.O. Box 386, 6700 AJ, Wageningen, The Netherlands.

Key Message: A chromosomal inversion associated with the tomato Ty - 2 gene for TYLCV resistance is the cause of severe suppression of recombination in a tomato Ty - 2 introgression line. Among tomato and its wild relatives inversions are often observed, which result in suppression of recombination. Such inversions hamper the transfer of important traits from a related species to the crop by introgression breeding. Suppression of recombination was reported for the TYLCV resistance gene, Ty-2, which has been introgressed in cultivated tomato (Solanum lycopersicum) from the wild relative S. habrochaites accession B6013. Ty-2 was mapped to a 300-kb region on the long arm of chromosome 11. The suppression of recombination in the Ty-2 region could be caused by chromosomal rearrangements in S. habrochaites compared with S. lycopersicum. With the aim of visualizing the genome structure of the Ty-2 region, we compared the draft de novo assembly of S. habrochaites accession LYC4 with the sequence of cultivated tomato ('Heinz'). Furthermore, using populations derived from intraspecific crosses of S. habrochaites accessions, the order of markers in the Ty-2 region was studied. Results showed the presence of an inversion of approximately 200 kb in the Ty-2 region when comparing S. lycopersicum and S. habrochaites. By sequencing a BAC clone from the Ty-2 introgression line, one inversion breakpoint was identified. Finally, the obtained results are discussed with respect to introgression breeding and the importance of a priori de novo sequencing of the species involved.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00122-015-2561-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4572051PMC
October 2015

Assessing the genetic variation of - and - alleles conferring resistance to tomato yellow leaf curl virus in a broad tomato germplasm.

Mol Breed 2015 26;35(6):132. Epub 2015 May 26.

Wageningen UR Plant Breeding, Wageningen University and Research Centre, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.

Tomato yellow leaf curl virus (TYLCV) hampers tomato production worldwide. Our previous studies have focussed on mapping and ultimately cloning of the TYLCV resistance genes - and -. Both genes are derived from and were shown to be allelic. They code for an RNA-dependent RNA polymerase (RDR) belonging to the RDRγ type defined by a DFDGD catalytic domain. In this study, we first fine-mapped the TYLCV resistance in LA1932, LA1960 and LA1971. Results showed that chromosomal intervals of the causal genes in these TYLCV-resistant accessions overlap and cover the region where -- is located. Further, virus-induced gene silencing was used to silence -/- in tomato lines carrying TYLCV resistance introgressed from LA1932, LA1938 and LA1971. Results showed that silencing -- compromised the resistance in lines derived from LA1932 and LA1938. The LA1971-derived material remained resistant upon silencing --. Further, we studied the allelic variation of the -- gene by examining cDNA sequences from nine -derived lines/accessions and more than 80 tomato cultivars, landraces and accessions of related wild species. The DFDGD catalytic domain of the -- gene is conserved among all tomato lines and species analysed. In addition, the 12 base pair insertion at the 5-prime part of the -- gene was found not to be specific for the TYLCV resistance allele. However, compared with the susceptible - allele, the -- allele is characterized by three specific amino acids shared by seven TYLCV-resistant accessions or derived lines. Thus, ---specific markers can be developed based on these polymorphisms. Elevated transcript levels were observed for all tested alleles (both - and - alleles), demonstrating that elevated expression level is not a good selection criterion for a functional -- allele.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11032-015-0329-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4442973PMC
May 2015

Identification of candidate MLO powdery mildew susceptibility genes in cultivated Solanaceae and functional characterization of tobacco NtMLO1.

Transgenic Res 2015 Oct 7;24(5):847-58. Epub 2015 May 7.

Laboratory of Plant Breeding, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.

Specific homologs of the plant Mildew Locus O (MLO) gene family act as susceptibility factors towards the powdery mildew (PM) fungal disease, causing significant economic losses in agricultural settings. Thus, in order to obtain PM resistant phenotypes, a general breeding strategy has been proposed, based on the selective inactivation of MLO susceptibility genes across cultivated species. In this study, PCR-based methodologies were used in order to isolate MLO genes from cultivated solanaceous crops that are hosts for PM fungi, namely eggplant, potato and tobacco, which were named SmMLO1, StMLO1 and NtMLO1, respectively. Based on phylogenetic analysis and sequence alignment, these genes were predicted to be orthologs of tomato SlMLO1 and pepper CaMLO2, previously shown to be required for PM pathogenesis. Full-length sequence of the tobacco homolog NtMLO1 was used for a heterologous transgenic complementation assay, resulting in its characterization as a PM susceptibility gene. The same assay showed that a single nucleotide change in a mutated NtMLO1 allele leads to complete gene loss-of-function. Results here presented, also including a complete overview of the tobacco and potato MLO gene families, are valuable to study MLO gene evolution in Solanaceae and for molecular breeding approaches aimed at introducing PM resistance using strategies of reverse genetics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11248-015-9878-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4569668PMC
October 2015

Mapping in the era of sequencing: high density genotyping and its application for mapping TYLCV resistance in Solanum pimpinellifolium.

BMC Genomics 2014 Dec 20;15:1152. Epub 2014 Dec 20.

Wageningen UR Plant Breeding, Wageningen University & Research Centre, P,O, Box 386, Wageningen, AJ 6700, the Netherlands.

Background: A RIL population between Solanum lycopersicum cv. Moneymaker and S. pimpinellifolium G1.1554 was genotyped with a custom made SNP array. Additionally, a subset of the lines was genotyped by sequencing (GBS).

Results: A total of 1974 polymorphic SNPs were selected to develop a linkage map of 715 unique genetic loci. We generated plots for visualizing the recombination patterns of the population relating physical and genetic positions along the genome.This linkage map was used to identify two QTLs for TYLCV resistance which contained favourable alleles derived from S. pimpinellifolium. Further GBS was used to saturate regions of interest, and the mapping resolution of the two QTLs was improved. The analysis showed highest significance on Chromosome 11 close to the region of 51.3 Mb (qTy-p11) and another on Chromosome 3 near 46.5 Mb (qTy-p3). Furthermore, we explored the population using untargeted metabolic profiling, and the most significant differences between susceptible and resistant plants were mainly associated with sucrose and flavonoid glycosides.

Conclusions: The SNP information obtained from an array allowed a first QTL screening of our RIL population. With additional SNP data of a RILs subset, obtained through GBS, we were able to perform an in silico mapping improvement to further confirm regions associated with our trait of interest. With the combination of different ~ omics platforms we provide valuable insight into the genetics of S. pimpinellifolium-derived TYLCV resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2164-15-1152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4367842PMC
December 2014