CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew
Santillán Martínez, Miguel I. ; Bracuto, Valentina ; Koseoglou, Eleni ; Appiano, Michela ; Jacobsen, Evert ; Visser, Richard G.F. ; Wolters, Anne-Marie A. ; Bai, Yuling - \ 2020
BMC Plant Biology 20 (2020)1. - ISSN 1471-2229
CRISPR/Cas9 - PMR4 - Powdery mildew - Susceptibility gene - Targeted mutagenesis
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 P owdery M ildew R esistance 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.
Ty-1, a universal resistance gene against geminiviruses that is compromised by co-replication of a betasatellite
Voorburg, Corien M. ; Yan, Zhe ; Bergua-Vidal, Maria ; Wolters, Anne Marie A. ; Bai, Yuling ; Kormelink, Richard - \ 2020
Molecular Plant Pathology 21 (2020)2. - ISSN 1464-6722 - p. 160 - 172.
beet curly top virus - betasatellite - geminivirus - resistance - RNA interference - tomato yellow leaf curl virus - Ty-1
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.
CRISPR/Cas inactivation of RECQ4 increases homeologous crossovers in an interspecific tomato hybrid
Maagd, Ruud A. de; Loonen, Annelies ; Chouaref, Jihed ; Pelé, Alexandre ; Meijer-Dekens, Fien ; Fransz, Paul ; Bai, Yuling - \ 2020
Plant Biotechnology Journal 18 (2020)3. - ISSN 1467-7644 - p. 805 - 813.
class II crossover pathway - interspecific crosses - introgression breeding - meiosis - RECQ4
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.
Breeding Has Increased the Diversity of Cultivated Tomato in The Netherlands
Schouten, Henk J. ; Tikunov, Yury ; Verkerke, Wouter ; Finkers, Richard ; Bovy, Arnaud ; Bai, Yuling ; Visser, Richard G.F. - \ 2019
Frontiers in Plant Science 10 (2019). - ISSN 1664-462X
breeding - diversity - introgressions - metabolomics - tomato varieties
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 Wasserbomben (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.
Effects of iron, calcium, and organic matter on phosphorus behavior in fluvo-aquic soil: farmland investigation and aging experiments
Ma, Yuling ; Ma, Jie ; Peng, Hao ; Weng, Liping ; Chen, Yali ; Li, Yongtao - \ 2019
Journal of Soils and Sediments 19 (2019)12. - ISSN 1439-0108 - p. 3994 - 4004.
Calcium carbonate - Fe mineral - Fluvo-aquic soil - Organic fertilizer - Phosphorus fractions - Phosphorus immobilization
Purpose: Excessive fertilization has led to a high risk of phosphorus (P) leaching and related problems in the North China Plain, where the most typical cropland soil is fluvo-aquic soil. The main factors controlling environmental P behavior and the acting time sequence of these factors in soil after long-term P fertilizer application have not been well recognized. A clear understanding is essential for effective P management. Materials and methods: Effects of Fe minerals, calcium carbonate, and organic matter (OM) on P immobilization in fluvo-aquic soil were studied systematically through farmland investigation and aging experiments. Results and discussion: Phosphorus associated with Ca was the primary fraction in fluvo-aquic soil. Even though there was no significant correlation between the total contents of P and Ca in soils, formation of P-Ca phases facilitated by Ca2+ in soil solution was a mechanism of P retention when soil received excess P fertilizer. Positive correlations between the contents of P and Fe and total organic carbon (TOC) indicate that Fe minerals and OM have significant effects on P immobilization. Through the aging experiments, P was found to primarily adsorb on goethite and gradually forms Ca-P compounds. Organic fertilizer caused P release and inhibition of P adsorption in the initial stages; however, OM derived from organic fertilizer might facilitate P immobilization in the long term through the formation of a P-Ca-OM complex. Conclusions: Although superfluous application of P fertilizers leads to the gradual formation of Ca-P in fluvo-aquic soils, there is still a risk of P loss because P is not immediately adsorbed by Fe minerals. Moreover, application of organic fertilizers increases the risk of P loss. These results provide an important scientific basis for initiating P management policies for fluvo-aquic soils.
Resistance to tomato yellow leaf curl virus in tomato germplasm
Yan, Zhe ; Pérez-de-Castro, Ana ; Díez, Maria J. ; Hutton, Samuel F. ; Visser, Richard G.F. ; Wolters, Anne-Marie A. ; Bai, Yuling ; Li, Junming - \ 2018
Frontiers in Plant Science 9 (2018). - ISSN 1664-462X
Begomovirus - Resistance - S. chilense - S. peruvianum - Solanum lycopersicum - Tomato - TYLCV
Tomato yellow leaf curl virus (TYLCV) is a virus species causing epidemics in tomato (Solanum lycopersicum) 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 S. arcanum, 43 from S. chilense, 1 from S. chmielewskii, 28 from S. corneliomulleri, 5 from S. habrochaites, 4 from S. huaylasense, 2 from S. neorickii, 1 from S. pennellii, 39 from S. peruvianum, and 1 from S. pimpinellifolium. Most of the screened S. chilense accessions remained symptomless. Many symptomless accessions were also identified in S. arcanum, S. corneliomulleri, and S. peruvianum. A large number of S. pimpinellifolium accessions were screened. However, almost all of the tested accessions showed TYLCD symptoms. Further, we studied allelic variation of the Ty-1/Ty-3 gene in few S. chilense 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 Ty-genes in TYLCV-resistant accessions.
Tomato disease resistances in the post-genomics era
Bai, Yuling ; Yan, Zhe ; Moriones, E. ; Fernández-Muñoz, R. - \ 2018
In: Proceedings of the 5th International Symposium on Tomato Diseases. - International Society for Horticultural Science (Acta Horticulturae ) - ISBN 9789462612037 - p. 1 - 17.
CRISPR/CAS9 - Effector target - Effector-assisted R gene identification - Gene editing - Mutagenesis - Recessive resistance - Resilience to combined stresses - TILLING
Disease in tomato (Solanum lycopersicum) can be caused by many pathogenic organisms, including cellular pathogens (e.g., fungi, bacteria, phytoplasmas, oomycetes and nematodes) and non-cellular pathogens (e.g., viruses and viroids). To respond to pathogen attack, tomato plants, like other sessile organisms, have developed an immune system, where pathogen effectors and plant receptor proteins (e.g., resistance proteins) play a central role. With advances in the genomics era, our understanding of plant-pathogen interactions is evolving rapidly. For example, pathogen genomics has allowed a genome-wide study on the structure, function and evolution of effectors in pathogen genomes. So-called effectoromics offers a high-throughput functional approach to study effector-associated plant genes such as resistance (R) genes and susceptibility (S) genes. In tomato, “genome to germplasms” is facilitating a genome dimension to the exploration of plant diversity in resistance by sequencing and re-sequencing of genomes of available germplasm resources. Together with this breakthrough and powerful techniques for genome editing, novel strategies are being developed for breeding tomatoes with durable resistance to pathogens. Using examples of several tomato diseases, this review focuses on (1) layers of plant immune system, (2) the exploitation of plant S genes in resistance breeding, (3) rapid identification of R and S genes, and (4) novel routes for durable resistance to pathogens. Finally, the topic of breeding for resilience to combined biotic and abiotic stresses is discussed based on our results, which show extensive crosstalk between loci/pathways for resistance to pathogens and tolerance to abiotic stresses.
The role of tomato WRKY genes in plant responses to combined abiotic and biotic stresses
Bai, Yuling ; Sunarti, Sri ; Kissoudis, Christos ; Visser, Richard G.F. ; Linden, C.G. van der - \ 2018
Frontiers in Plant Science 9 (2018). - ISSN 1664-462X
Abiotic stress - Biotic stress - Combined stresses - Disease resistance - Effector-triggered immunity (ETI) - PAMP-triggered immunity (PTI)
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 WRKY genes identified. Here we review recent progress on functions of these tomato WRKY 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 WRKY genes that play a role in plant responses to a combination of abiotic and biotic stresses.
Neglected environmental health impacts of China's supply-side structural reform
Zhang, Wei ; Zhang, Lei ; Li, Ying ; Tian, Yuling ; Li, Xiaoran ; Zhang, Xue ; Mol, Arthur P.J. ; Sonnenfeld, David A. ; Liu, Jianguo ; Ping, Zeyu ; Chen, Long - \ 2018
Environment International 115 (2018). - ISSN 0160-4120 - p. 97 - 103.
Air quality - Environmental policy - Health effects of air pollution - Supply-side structural reform - Transregional distribution
“Supply-side structural reform” (SSSR) has been the most important ongoing economic reform in China since 2015, but its important environmental health effects have not been properly assessed. The present study addresses that gap by focusing on reduction of overcapacity in the coal, steel, and iron sectors, combined with reduction of emissions of sulfur dioxide (SO2), nitrogen oxide (NOx), and fine particulate matter (PM2.5), and projecting resultant effects on air quality and public health across cities and regions in China. Modeling results indicate that effects on air quality and public health are visible and distributed unevenly across the country. This assessment provides quantitative evidence supporting projections of the transregional distribution of such effects. Such uneven transregional distribution complicates management of air quality and health risks in China. The results challenge approaches that rely solely on cities to improve air quality. The article concludes with suggestions on how to integrate SSSR measures with cities’ air quality improvement attainment planning and management performance evaluation.
Plant behaviour under combined stress : tomato responses to combined salinity and pathogen stress
Bai, Yuling ; Kissoudis, Christos ; Yan, Zhe ; Visser, Richard G.F. ; Linden, Gerard van der - \ 2018
The Plant Journal 93 (2018)4. - ISSN 0960-7412 - p. 781 - 793.
combined biotic and abiotic stresses - plant disease - resistance gene - salinity stress - stress interaction - stress tolerance
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.
Trichomes: interaction sites of tomato leaves with biotrophic powdery mildew pathogens
Suzuki, Tomoko ; Murakami, Tomoe ; Takizumi, Yoshihiro ; Ishimaru, Hiroyuki ; Kudo, Daiki ; Takikawa, Yoshihiro ; Matsuda, Yoshinori ; Kakutani, Koji ; Bai, Yuling ; Nonomura, Teruo - \ 2018
European Journal of Plant Pathology 150 (2018)1. - ISSN 0929-1873 - p. 115 - 125.
Delayed cell death - Erysiphe trifoliorum - Hypersensitive cell death - Papilla formation - Podosphaera xanthii - Pseudoidium neolycopersici
The present study aimed to explore the possibility of using the type I trichomes of tomato (Solanum lycopersicum) to monitor the infection processes of powdery mildews by microscopy. Individual trichome cells of two tomato genotypes were inoculated with pathogenic and non-pathogenic powdery mildew species, Pseudoidium neolycopersici, Erysiphe trifoliorum and Podosphaera xanthii. On the trichome cells of the tomato cultivar Moneymaker, hyphae of the pathogenic Pseudoidium neolycopersici (isolates KTP-03 and KTP-04) grew vigorously; whereas hyphal growth of the non-pathogenic Erysiphe trifoliorum and Podosphaera xanthii ceased after appressorium formation, which was associated with papilla formation and hypersensitive cell death, respectively. Similar infection processes of the tested powdery mildews were seen in Moneymaker epidermal cells. Therefore, tomato trichomes are suitable for analysing, at individual cell level, the infection processes of different pathotypes of powdery mildews and for observing the cytological responses of plants by non-pathogenic powdery mildews. On the other hand, it was observed that both isolates KTP-03 and KTP-04 failed to produce conidiophores on the hyphae elongating on Moneymaker trichomes. Similarly, no conidiophores were produced on the hyphae elongating on trichomes of Solanum peruvianum LA2172, which is resistant to KTP-03 and susceptible to KTP-04. Interestingly, delayed cell death occurred in LA2172 epidermal cells, which were attacked by KTP-03 hyphae elongating from trichomes and conidiophores were formed on new hyphae growing from the leaf epidermal cells. Thus, leaf trichomes and epidermal cells of the wild tomato species LA2172 reacted differently to the avirulent isolate KTP-03.
Functional characterization of a syntaxin involved in tomato (Solanum lycopersicum) resistance against powdery mildew
Bracuto, Valentina ; Appiano, Michela ; Zheng, Zheng ; Wolters, Anne-Marie A. ; Yan, Zhe ; Ricciardi, Luigi ; Visser, Richard G.F. ; Pavan, Stefano ; Bai, Yuling - \ 2017
Frontiers in Plant Science 8 (2017). - ISSN 1664-462X - 10 p.
Blumeria graminis f. sp. hordei - Mlo resistance - Non-host resistance - Oidium neolycopersici - Tomato syntaxins
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 SlPEN1a and SlPEN1b, are closely related to AtPEN1. RNAi-based silencing of SlPEN1a in a tomato line carrying a loss-of-function mutation of the susceptibility gene SlMLO1 led to compromised resistance toward the tomato powdery mildew fungus Oidium neolycopersici. Moreover, it resulted in a significant increase in the penetration rate of the non-adapted powdery mildew fungus Blumeria graminis f. sp. hordei. 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.
Key components of different plant defense pathways are dispensable for powdery mildew resistance of the arabidopsis mlo2 mlo6 mlo12 triple mutant
Kuhn, Hannah ; Lorek, Justine ; Kwaaitaal, Mark ; Consonni, Chiara ; Becker, Katia ; Micali, Cristina ; Themaat, Emiel Ver Loren Van; Bednarek, Paweł ; Raaymakers, Tom M. ; Appiano, Michela ; Bai, Yuling ; Meldau, Dorothea ; Baum, Stephani ; Conrath, Uwe ; Feussner, Ivo ; Panstruga, Ralph - \ 2017
Frontiers in Plant Science 8 (2017). - ISSN 1664-462X
Camalexin - Indole glucosinolates - Jasmonic acid - Microarray analysis - MLO - Plant defense - Powdery mildew - Tryptophan
Loss of function mutations of particular plant MILDEW RESISTANCE LOCUS O (MLO) 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 Arabidopsis thaliana mlo2 mlo6 mlo12 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 mlo2 single mutants. Comparative microarray-based transcriptome analysis of mlo2 mlo6 mlo12 mutants and wild type plants upon Golovinomyces orontii 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 mlo2 mlo6 mlo12 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.
Functional characterization of cucumber (Cucumis sativus L.) Clade V MLO genes
Berg, Jeroen A. ; Appiano, Michela ; Bijsterbosch, Gerard ; Visser, Richard G.F. ; Schouten, Henk J. ; Bai, Yuling - \ 2017
BMC Plant Biology 17 (2017)1. - ISSN 1471-2229
Cucumber (Cucumis sativus L.) - Gene expression - MLO - Powdery mildew - Susceptibility genes
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.
Functional characterization of the powdery mildew susceptibility gene SmMLO1 in eggplant (Solanum melongena L.)
Bracuto, Valentina ; Appiano, Michela ; Ricciardi, Luigi ; Göl, Deniz ; Visser, Richard G.F. ; Bai, Yuling ; Pavan, Stefano - \ 2017
Transgenic Research 26 (2017)3. - ISSN 0962-8819 - p. 323 - 330.
Eggplant - MLO - Plant breeding - Powdery mildew - Resistance
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.
Ethylene and abscisic acid signaling pathways differentially influence tomato resistance to combined powdery mildew and salt stress
Kissoudis, Christos ; Seifi, Alireza ; Yan, Zhe ; Islam, Tanjimul ; Schoot, Hanneke van der; Wiel, Clemens C.M. van de; Visser, Richard G.F. ; Linden, C.G. van der; Bai, Yuling - \ 2017
Frontiers in Plant Science 7 (2017). - ISSN 1664-462X
Abscisic acid - Callose - Cell death - Chitinase - ROS burst - Senescence
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 Ol-1, ol-2, and Ol-4 loci, conferring resistance to tomato powdery mildew (PM) caused by Oidium neolycopersici, to combined PM and salt stress. These NILs were crossed with the notabilis (ABAdeficient), defenceless1 (JA-deficient), and epinastic (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 epinastic mutation resulted in breakdown of resistance in NILOl- 1 and NIL-ol-2. This was accompanied by reduced callose deposition, and was more pronounced under combined salt stress. The notabilis mutation resulted in H2O2 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 notabilis 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.
Frequency of a natural truncated allele of MdMLO19 in the germplasm of Malus domestica
Pessina, Stefano ; Palmieri, Luisa ; Bianco, Luca ; Gassmann, Jennifer ; De Weg, Eric Van; Visser, Richard G.F. ; Magnago, Pierluigi ; Schouten, Henk J. ; Bai, Yuling ; Riccardo Velasco, R. ; Malnoy, Mickael - \ 2017
Molecular Breeding 37 (2017)1. - ISSN 1380-3743
Podosphaera leucotricha 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 MLO gene family that are responsible for PM susceptibility (S-genes). In apple, the knockdown of MdMLO19 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 MdMLO19 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 MLO genes (MdMLO5, MdMLO7, MdMLO11, MdMLO18, and MdMLO19), 127 of which were located in exons. The T-1201 insertion of a single nucleotide in MdMLO19 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 mlo allele has been found
Susceptibility pays off: insights into the mlo-based powdery mildew resistance
Appiano, Michela - \ 2016
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Yuling Bai; Anne-Marie Wolters. - Wageningen : Wageningen University - ISBN 9789462579484 - 265
solanum lycopersicum - tomatoes - disease resistance - susceptibility - oidium neolycopersici - genes - gene expression - genomics - molecular breeding - plant breeding - solanum lycopersicum - tomaten - ziekteresistentie - vatbaarheid - oidium neolycopersici - genen - genexpressie - genomica - moleculaire veredeling - plantenveredeling
Powdery mildew (PM) is a worldwide-occurring plant disease caused by ascomycete fungi of the order Erysiphales. A conspicuous number of plant species are susceptible to this disease, the occurrence of which is increasing due to the influence of climate change. Symptoms are easy to recognize by the powdery whitish fungal structures growing on the surface of plant organs. Severe infections cause significant losses in crops, such as tomato, cucumber and wheat, as well as in ornamentals, like rose and petunia. Accordingly, breeding crops with a robust immunity to this disease is of great economic importance.
A significant step in this direction was the discovery of mlo (mildew locus o) mutant alleles of the barley HvMlo gene, which are responsible for the non-race specific resistance to the barley PM pathogen, Blumeria graminis f.sp. hordei (Bgh). During the years, this recessively inherited resistance was observed to be durable, contrary to the short life-span of resistances conferred by dominant resistance (R-) genes used in barley breeding programs. Studies on the histological mechanisms of the mlo-based resistance showed that the PM pathogen was stopped during penetration of the cell wall by the formation of a papilla. This structure prevents the formation of the feeding structure of the pathogen, called a haustorium.
After sequencing many plant genomes, we are discovering that MLO genes are not only typical of this cereal, but are ubiquitously present in higher plant species in multiple copies per species, forming a gene family. The impairment of some members of a number of ever increasing plant species lead to broad-spectrum resistance towards their adapted PM pathogens. For example, in tomato the ol-2 gene, naturally harbored by the cherry tomato Solanum lycopersicum var. cerasiforme, represents the loss-of-function allele of the SlMLO1 gene, conferring resistance to the PM pathogen Oidium neolycopersici (On). Consequently, the use of mlo mutants represents a suitable alternative to the classical use of R-genes in breeding programs.
In Chapter 2, we describe the in silico identification of the complete tomato SlMLO gene family using the available information in the SOL genomic network database. In total, 16 tomato SlMLO members were cloned from leaf, root, flower and fruit of the susceptible tomato cv. Moneymaker to confirm the sequences retrieved from the database and to verify their actual expression in these tissues. We observed the presence of various types of splicing variants, although their possible functional meaning has not been investigated. Motif analyses of each of the translated protein sequences and phylogenetic studies highlighted, on one hand, amino acid stretches that characterize the whole MLO family, and, on the other hand, stretches conserved in MLO homologs that are phylogenetically related. Following a gene expression study upon On inoculation, we identified members of the SlMLO family that are upregulated few hours after pathogen challenge. Except SlMLO1, none of the three newly identified homologs in clade V, thus phylogenetically close to SlMLO1, are induced. Interestingly, two homologs, each found in different clades, are upregulated similarly to SlMLO1. Using an RNAi approach, we silenced the additional clade V-SlMLO homologs, namely SlMLO3, SlMLO5 and SlMLO8, to investigate their possible role in PM resistance. We observed that none of these homologs if individually silenced, leads to PM resistance. However, if SlMLO5 and SlMLO8 are silenced together with SlMLO1, a significantly higher level of resistance is achieved compared to plants carrying the ol-2 allele. The role of SlMLO3 could not be verified. We, therefore, concluded that there are three SlMLO genes in tomato unevenly contributing to the PM disease, of which SlMLO1 has a major role.
Chapter 3 focuses on the components of the tomato mlo-based resistance. In Arabidopsis, it is known that four members of the SNARE protein family, involved in membrane fusion, are involved in mlo-based resistance. In this chapter, we focused on the identification of tomato homologs of the Arabidopsis syntaxin PEN1 (AtSYP121). Among the group of syntaxins identified in tomato, two were closely related to each other and also to AtPEN1, denominated SlPEN1a and SlPEN1b. Another Arabidopsis syntaxin that shows a high level of homology with PEN1, called SYP122, was also found to group together with the newly identified SlPEN1 genes. However, the role of SYP122 in plant immunity was not shown in literature. After obtaining individual silencing RNAi constructs, we transformed the resistant ol-2 line, and we challenged the obtained transformants with the adapted PM On, and the non-adapted Bgh. Interestingly, we observed a significant On growth and an enhanced Bgh cell entry only in SlPEN1a silenced plants but not in SlPEN1b silenced ones. We performed a protein alignment of tomato and Arabidopsis functional and non-functional PEN sequences. The presence of three differently conserved non-synonymous amino-acid substitutions is hypothesised to be responsible for the specialization in plant immune function.
In Chapter 4 and Chapter 5, we build up a body of evidence pointing to the fact that the function of the MLO susceptibility genes is highly conserved between monocot and dicot plant species.
In Chapter 4 we started by identifying and functionally characterizing two new MLO genes of Solanaceous crops affected by the PM disease, tobacco (Nicotiana tabacum) and eggplant (Solanum melongena). We named them NtMLO1 and SmMLO1 in the respective species, as they are the closest homologs to tomato SlMLO1. By overexpressing these genes in the resistant ol-2 line, we obtained transgenic plants that were susceptible to the PM pathogen On. This finding demonstrates that both heterologous MLO proteins can rescue the function of the impaired ol-2 allele in tomato. In addition, we found in tobacco NtMLO1 an amino acid (Q198) of critical importance for the susceptibility function of this protein.
In Chapter 5, we used the same approach adopted in Chapter 4 to show that other MLO proteins of more distant dicot species, like pea PsMLO1, can rescue the loss-of-function of the tomato ol-2 allele. And finally, we stretched this concept also to monocot MLO proteins, using barley HvMlo. While performing these experiments, we could verify that the function of the monocot and dicot susceptibility MLO proteins does not rely on the presence of class-specific conservation. The latter can be the reason for the phylogenetic divergence, placing monocot MLO proteins in clade IV and dicot MLO proteins in clade V of the phylogenetic MLO tree. However, functional conservation might depend on crucial shared amino acids of clade IV and V MLO proteins. Therefore, we also conducted a codon-based evolutionary analysis that resulted in the identification of 130 codons under negative selection, thus strongly maintained during evolution.
In Chapter 6 we introduce the PM disease in cucumber caused by Podosphaera xanthii (Px). We cloned the candidate susceptibility gene for PM in cucumber, CsaMLO8, from susceptible and resistant genotypes. The latter was described as an advanced cucumber breeding line characterized by hypocotyl resistance. In this line, we found the presence of aberrant splicing variants of the CsaMLO8 mRNA due to the insertion in its corresponding genomic region of a Class LTR retrotransposon. Heterologous expression of the wild-type cucumber allele in the tomato ol-2 line restored its PM susceptibility, while the heterologous expression of the aberrant protein variant failed to do so. This finding confirms that the resistance of the advanced cucumber breeding line is due to the disruption of the coding region of this gene. We also showed that the expression of CsaMLO8 in the susceptible genotype is induced by Px in hypocotyl tissue, but not in cotyledon or leaf. Finally, by examination of the resequencing data of a collection of 115 cucumber accessions, we found the presence of the TE-containing allele in 31 of them among which a wild cucumber accession that might have been used in breeding programs to obtain resistance to the PM disease in cucumber.
In Chapter 7 a novel loss-of-function allele of the SlMLO1 gene is described, designated m200. This allele was found in a resistant plant (M200) from a mutagenized tomato Micro-Tom (MT) population obtained with the chemical mutagen ethyl methanesulfonate (EMS). The m200 mutation corresponds to a nucleotide transversion (T à A) which results in a premature stop codon. The length of the predicted SlMLO1 protein in the M200 plant is only 21 amino acids, thus much shorter than the predicted protein of the previously described ol-2 allele, consisting of 200 amino acids. Thanks to the development of a High-Resolution Melting (HRM) marker designed to detect the m200 mutation, we observed that this allele confers recessively inherited resistance in backcross populations of the resistant M200 plant with MT and Moneymaker. Histological study showed that the resistance of the m200 mutant is associated with papilla formation. Finally, we compared the rate of On penetration in epidermal cells of m200 plants with the one of plants carrying the ol-2 allele and the transgenic plants in which multiple SlMLO homologs were silenced, generated in Chapter 2.
Ultimately, in Chapter 8 the results of the previous chapters are discussed in the context of 1) practical applications in breeding programs aimed at introducing the mlo-based resistance in new crops, 2) possible research aimed at unraveling the function of the MLO protein and 3) the role of other SNARE proteins.
Responses to combined abiotic and biotic stress in tomato are governed by stress intensity and resistance mechanism
Kissoudis, Christos ; Sri Sunarti, Sri ; De Wiel, Clemens Van; Visser, Richard G.F. ; Linden, Gerard van der; Bai, Yuling - \ 2016
Journal of Experimental Botany 67 (2016)17. - ISSN 0022-0957 - p. 5119 - 5132.
Callose - cell death - ethylene - invertase - R-gene resistance - stress severity
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.
Silencing of six susceptibility genes results in potato late blight resistance
Sun, Kaile ; Wolters, Anne-Marie A. ; Vossen, Jack H. ; Rouwet, Maarten E. ; Loonen, Annelies E.H.M. ; Jacobsen, Evert ; Visser, Richard G.F. ; Bai, Yuling - \ 2016
Transgenic Research 25 (2016)5. - ISSN 0962-8819 - p. 731 - 742.
Late blight - Potato - Resistance - RNAi - Susceptibility gene
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. thalianaS-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.