Unveiling and deploying durability of late blight resistance in potato : from natural stacking to cisgenic stacking
The potato, which receives an increased attention as a food crop, has long been in threats from the oomycete Phytophthora infestans, the causal agent of late blight. This disease still remains the most important constraint in potato producing regions of the world. It might cause the complete destruction of the foliage and tubers of potato if meteorological conditions are conducive to the onset and spread of late blight epidemics. Although fungicides applications provide sufficient levels of late blight control, they impose high input costs to the farmer, are detrimental to human and environment and increase the capacity of the pathogen to develop resistance to the active ingredients of fungicides applied. The increased genetic diversity in P. infestanspopulations due to sexual recombination between two mating types in many parts of the world and the emergence of fungicide resistant strains poses the necessity to develop potatoes that possess high levels of durable resistance as an alternative to the use of fungicides. Clones MaR8 and MaR9 from the Mastenbroek differential set, used to assess virulence towards Rgenes, have been known for their strong resistance to P. infestans. This also holds for cultivar Sarpo Mira which has retained resistance in the field over several years without fungicide applications. Uncovering genetic basis of such, partly naturally-formed, late blight resistance is the prerequisite for the implementation of durable resistance in a breeding scheme. In this study, MaR8, MaR9and cv. Sarpo Mira were used as plant materials for unveiling durability of late blight resistance in potato. First, F1 mapping populations from crosses between these resistant materials with susceptible parents were assessed for late blight resistance in field trials and in detached leaf assays (DLA) after inoculation with an incompatible P. infestans isolate IPO-C. A 1:1 segregation of resistance and susceptibility was observed in the MaR8derived-F1 population in field trials, but not in detached leaf assays. NBS profiling and Rgene cluster directed profiling (CDP), followed by marker landing in the newly sequenced potato genome, referred to as “anchored scaffold approach”, led to the mapping of R8at a new locus on chromosome IX rather than on chromosome XI, the previously suggested chromosomal position (Chapter 2). The Rgene mediated resistance reaction in potato is a consequence of an (in)direct interaction between the pathogen Avrand host Rgene product that leads to a hypersensitive cell death (HR). We screened a wide collection of RXLR effectors of P. infestansfor eliciting cell death in the differential potato MaR8 by agroinfiltration (Chapter 3). R8-specific cell death to one effector PITG_07558, termed AVR8, co-segregated with the R8-mediated resistance to P. infestansisolate IPO-C in a F1 population. From the notion that Avr8is identical to effector AvrSmira2that was previously found to associate with field resistance in cultivar Sarpo Mira, we performed genetic mapping studies in a Sarpo Mira-based F1 population and indeed Rpi-Smira2localized in the R8locus. To investigate the geographical and phylogenetic origin of R8in the Solanumgene pool, we conducted functional screens for AVR8 responsiveness in 98 wild genotypes (72 accessions of 40 species) of Solanumsection Petota. We identified twelve AVR8 responding Solanum accessions originating both from Central and South America. Interestingly, our study involving late blight resistance from the differential plant MaR9described that it is near the R8 locus on chromosome IX (Chapter 4). An integrated approach combining 1. a Rgene ”de-stacking” approach using Rgene specific marker analysis and effector responses, 2. the whole plant climate cell assay, and 3. CDP profiling enabled a clear picture for the presence of two closely linked genes, termed R9aand R9b. It was shown that R9alocates in a Tm-22 cluster of NB-LRR genes and, most likely will be a member of the Tm-22 Rgene family (Chapter 4). The identified fully co-segregating Tm-2 likeCDP markers were used to select the R9agene-containing BAC clone, demonstrating the possibility of BAC landing by marker saturation in the targeted chromosomal regions (Chapter 5). For cloning R9agene, a bacterial artificial chromosome (BAC) library derived from the differential plant MaR9, was screened with co-segregating Rgene CDP markers whereby two overlapping BAC clones carrying CDP markers were obtained. Sequence annotation of the complete insert of these BAC clones identified the presence of two complete Rgene analogs (RGA9.1 andRGA9.2) of the NB-LRR class in one BAC clone. Two RGAs, including their natural regulatory transcriptional elements, were subcloned by long-range PCR into a binary vector for plant transformation. After transformation, it was found that RGA9.1was able to complement the susceptible phenotype in cultivar Desiree. RGA9.1, now designated R9a,encodes a CC-NB-LRR protein of the Tm2 family, where the LRR consensus is only loosely fitted. Agroinfiltration-based effector screens for identifying the Avrgenes matching the R9agene was performed, leading to the discovery of Avrblb2 homologs which trigger R9amediated hypersensitivity in Nicotiana benthamiana (Chapter 5).Resistance profiling with 54 P. infestans isolates showed that MaR9 and S.xedinense accessions had similar resistance spectra as the Rpi-blb2containing cultivar Bionica. Transformation of potato with resistance genes and antibiotic resistance markers encounters consumers’ criticism. These criticisms are considerably less if only resistance genes from crossable species, and no antibiotic resistance selection marker is used. Genes deriving from crossable species are referred to as cisgenes. For the production of cisgenic potatoes with a broader resistance spectrum and potential durability, Agrobacterium-mediated marker free transformation and PCR selection of transformants was performed. This way four potato cultivars (Atlantic, Bintje, Potae9 and Doip1) were successfully transformed with a construct containing two cisgenic Rpigenes (Rpi-vnt1from Solanum venturiiand Rpi-sto1from Solanum stoloniferum) (Chapter 6). Resistance assays in untransformed varieties with five P. infestansisolates showed that cvs. Potae9 and Doip1 were already resistant to certain isolates. Single Rpigene containing transgenic plants for all 4 varieties were obtained and used as references. Marker free transformation with a construct containing two Rpi genes (cisgenesis) was compared to kanamycin assisted selection (transgenesis) in terms of regeneration and transformation frequency, vector backbone integration, and T-DNA copy number. In addition, the different time tracks to harvest regenerated shoots for the selection of PCR positive regenerants for one or both Rpi-genes were studied. Through further analyses involving phenotypic evaluations in the greenhouse, agroinfiltration of avirulence (Avr) genes and detached leaf assays, totally eight cisgenic plants were selected. Two cisgenic plants of cv. Altantic and four of cv. Bintje, were selected that showed broad spectrum late blight resistance due to the activity of both Rpigenes. Based on characterization of two cisgenic transformants of cv. Potae9, it was demonstrated that the existing late blight resistance spectrum has been broadened by adding the two Rpigenes. Finally, results from this study are discussed in terms of genetic and molecular mechanism of durability and cisgenic deployment to address the challenges of the durable resistant potato variety development (Chapter 7). We pursue possible options for durability in the nature of the Rgenes or their cognate Avrgenes. The comparative analysis of several features of available R-AVR pairs shows that major components for producing durability are the copy number variation in the P. infestansgenome and abundance of the Avrgene in different isolates. As a counterpart of such an Avrgene, potato Rgenes that display broad spectrum resistance and often have abundant functional homologs among various wild Solanumspecies could be optional for Rgene combinations providing durability. Multiple years’ on-site-monitoring of resistance spectrum in natural Rgene stacks demonstrates that stacking of several broad spectrum Rpigenes or even “defeated” Rgenes could sum up to high levels of resistance potentially capable to provide durability to commercial potato cultivars. Our data about acquirement of complementary resistance spectrum by cisgenic introduction of two broad spectrum resistance genes into cultivars support a first step into that direction.
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
| Subjects: | defence mechanisms, disease resistance, gene mapping, phytophthora infestans, plant pathogenic fungi, potatoes, solanum tuberosum, transgenic plants, aardappelen, genkartering, plantenziekteverwekkende schimmels, transgene planten, verdedigingsmechanismen, ziekteresistentie, |
| Online Access: | https://research.wur.nl/en/publications/unveiling-and-deploying-durability-of-late-blight-resistance-in-p |
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| Summary: | The potato, which receives an increased attention as a food crop, has long been in threats from the oomycete Phytophthora infestans, the causal agent of late blight. This disease still remains the most important constraint in potato producing regions of the world. It might cause the complete destruction of the foliage and tubers of potato if meteorological conditions are conducive to the onset and spread of late blight epidemics. Although fungicides applications provide sufficient levels of late blight control, they impose high input costs to the farmer, are detrimental to human and environment and increase the capacity of the pathogen to develop resistance to the active ingredients of fungicides applied. The increased genetic diversity in P. infestanspopulations due to sexual recombination between two mating types in many parts of the world and the emergence of fungicide resistant strains poses the necessity to develop potatoes that possess high levels of durable resistance as an alternative to the use of fungicides. Clones MaR8 and MaR9 from the Mastenbroek differential set, used to assess virulence towards Rgenes, have been known for their strong resistance to P. infestans. This also holds for cultivar Sarpo Mira which has retained resistance in the field over several years without fungicide applications. Uncovering genetic basis of such, partly naturally-formed, late blight resistance is the prerequisite for the implementation of durable resistance in a breeding scheme. In this study, MaR8, MaR9and cv. Sarpo Mira were used as plant materials for unveiling durability of late blight resistance in potato. First, F1 mapping populations from crosses between these resistant materials with susceptible parents were assessed for late blight resistance in field trials and in detached leaf assays (DLA) after inoculation with an incompatible P. infestans isolate IPO-C. A 1:1 segregation of resistance and susceptibility was observed in the MaR8derived-F1 population in field trials, but not in detached leaf assays. NBS profiling and Rgene cluster directed profiling (CDP), followed by marker landing in the newly sequenced potato genome, referred to as “anchored scaffold approach”, led to the mapping of R8at a new locus on chromosome IX rather than on chromosome XI, the previously suggested chromosomal position (Chapter 2). The Rgene mediated resistance reaction in potato is a consequence of an (in)direct interaction between the pathogen Avrand host Rgene product that leads to a hypersensitive cell death (HR). We screened a wide collection of RXLR effectors of P. infestansfor eliciting cell death in the differential potato MaR8 by agroinfiltration (Chapter 3). R8-specific cell death to one effector PITG_07558, termed AVR8, co-segregated with the R8-mediated resistance to P. infestansisolate IPO-C in a F1 population. From the notion that Avr8is identical to effector AvrSmira2that was previously found to associate with field resistance in cultivar Sarpo Mira, we performed genetic mapping studies in a Sarpo Mira-based F1 population and indeed Rpi-Smira2localized in the R8locus. To investigate the geographical and phylogenetic origin of R8in the Solanumgene pool, we conducted functional screens for AVR8 responsiveness in 98 wild genotypes (72 accessions of 40 species) of Solanumsection Petota. We identified twelve AVR8 responding Solanum accessions originating both from Central and South America. Interestingly, our study involving late blight resistance from the differential plant MaR9described that it is near the R8 locus on chromosome IX (Chapter 4). An integrated approach combining 1. a Rgene ”de-stacking” approach using Rgene specific marker analysis and effector responses, 2. the whole plant climate cell assay, and 3. CDP profiling enabled a clear picture for the presence of two closely linked genes, termed R9aand R9b. It was shown that R9alocates in a Tm-22 cluster of NB-LRR genes and, most likely will be a member of the Tm-22 Rgene family (Chapter 4). The identified fully co-segregating Tm-2 likeCDP markers were used to select the R9agene-containing BAC clone, demonstrating the possibility of BAC landing by marker saturation in the targeted chromosomal regions (Chapter 5). For cloning R9agene, a bacterial artificial chromosome (BAC) library derived from the differential plant MaR9, was screened with co-segregating Rgene CDP markers whereby two overlapping BAC clones carrying CDP markers were obtained. Sequence annotation of the complete insert of these BAC clones identified the presence of two complete Rgene analogs (RGA9.1 andRGA9.2) of the NB-LRR class in one BAC clone. Two RGAs, including their natural regulatory transcriptional elements, were subcloned by long-range PCR into a binary vector for plant transformation. After transformation, it was found that RGA9.1was able to complement the susceptible phenotype in cultivar Desiree. RGA9.1, now designated R9a,encodes a CC-NB-LRR protein of the Tm2 family, where the LRR consensus is only loosely fitted. Agroinfiltration-based effector screens for identifying the Avrgenes matching the R9agene was performed, leading to the discovery of Avrblb2 homologs which trigger R9amediated hypersensitivity in Nicotiana benthamiana (Chapter 5).Resistance profiling with 54 P. infestans isolates showed that MaR9 and S.xedinense accessions had similar resistance spectra as the Rpi-blb2containing cultivar Bionica. Transformation of potato with resistance genes and antibiotic resistance markers encounters consumers’ criticism. These criticisms are considerably less if only resistance genes from crossable species, and no antibiotic resistance selection marker is used. Genes deriving from crossable species are referred to as cisgenes. For the production of cisgenic potatoes with a broader resistance spectrum and potential durability, Agrobacterium-mediated marker free transformation and PCR selection of transformants was performed. This way four potato cultivars (Atlantic, Bintje, Potae9 and Doip1) were successfully transformed with a construct containing two cisgenic Rpigenes (Rpi-vnt1from Solanum venturiiand Rpi-sto1from Solanum stoloniferum) (Chapter 6). Resistance assays in untransformed varieties with five P. infestansisolates showed that cvs. Potae9 and Doip1 were already resistant to certain isolates. Single Rpigene containing transgenic plants for all 4 varieties were obtained and used as references. Marker free transformation with a construct containing two Rpi genes (cisgenesis) was compared to kanamycin assisted selection (transgenesis) in terms of regeneration and transformation frequency, vector backbone integration, and T-DNA copy number. In addition, the different time tracks to harvest regenerated shoots for the selection of PCR positive regenerants for one or both Rpi-genes were studied. Through further analyses involving phenotypic evaluations in the greenhouse, agroinfiltration of avirulence (Avr) genes and detached leaf assays, totally eight cisgenic plants were selected. Two cisgenic plants of cv. Altantic and four of cv. Bintje, were selected that showed broad spectrum late blight resistance due to the activity of both Rpigenes. Based on characterization of two cisgenic transformants of cv. Potae9, it was demonstrated that the existing late blight resistance spectrum has been broadened by adding the two Rpigenes. Finally, results from this study are discussed in terms of genetic and molecular mechanism of durability and cisgenic deployment to address the challenges of the durable resistant potato variety development (Chapter 7). We pursue possible options for durability in the nature of the Rgenes or their cognate Avrgenes. The comparative analysis of several features of available R-AVR pairs shows that major components for producing durability are the copy number variation in the P. infestansgenome and abundance of the Avrgene in different isolates. As a counterpart of such an Avrgene, potato Rgenes that display broad spectrum resistance and often have abundant functional homologs among various wild Solanumspecies could be optional for Rgene combinations providing durability. Multiple years’ on-site-monitoring of resistance spectrum in natural Rgene stacks demonstrates that stacking of several broad spectrum Rpigenes or even “defeated” Rgenes could sum up to high levels of resistance potentially capable to provide durability to commercial potato cultivars. Our data about acquirement of complementary resistance spectrum by cisgenic introduction of two broad spectrum resistance genes into cultivars support a first step into that direction. |
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