The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant-pathogen interactions
Karimi Jashni, M. ; Mehrabi, R. ; Collemare, J. ; Mesarich, C.H. ; Wit, P.J.G.M. de - \ 2015
Frontiers in Plant Science 6 (2015). - ISSN 1664-462X - 7 p.
cf-2-dependent disease resistance - extracellular serine-protease - l. enhances resistance - class iv chitinases - phytophthora-infestans - cladosporium-fulvum - proteolytic-enzymes - antifungal activity - gene-expression - tomato
Upon host penetration, fungal pathogens secrete a plethora of effectors to promote disease, including proteases that degrade plant antimicrobial proteins, and protease inhibitors (PIs) that inhibit plant proteases with antimicrobial activity. Conversely, plants secrete proteases and PIs to protect themselves against pathogens or to mediate recognition of pathogen proteases and PIs, which leads to induction of defense responses. Many examples of proteases and PIs mediating effector-triggered immunity in host plants have been reported in the literature, but little is known about their role in compromising basal defense responses induced by microbe-associated molecular patterns. Recently, several reports appeared in literature on secreted fungal proteases that modify or degrade pathogenesis-related proteins, including plant chitinases or PIs that compromise their activities. This prompted us to review the recent advances on proteases and PIs involved in fungal virulence and plant defense. Proteases and PIs from plants and their fungal pathogens play an important role in the arms race between plants and pathogens, which has resulted in co-evolutionary diversification and adaptation shaping pathogen lifestyles.
Genome mining and metabolic profiling of the rhizosphere bacterium Pseudomonas sp. SH-C52 for antimicrobial compounds
Voort, M. van der; Meijer, H.J.G. ; Schmidt, Y. ; Watrous, J. ; Dekkers, E. ; Mendes, R. ; Dorrestein, P.C. ; Gross, H. ; Raaijmakers, J.M. - \ 2015
Frontiers in Microbiology 6 (2015). - ISSN 1664-302X - 14 p.
ii secretion system - phytophthora-infestans - biological-activity - functional-analysis - mass-spectrometry - natural functions - phospholipase-d - corrugata - biocontrol - lipopeptides
The plant microbiome represents an enormous untapped resource for discovering novel genes and bioactive compounds. Previously, we isolated Pseudomonas sp. SH-C52 from the rhizosphere of sugar beet plants grown in a soil suppressive to the fungal pathogen Rhizoctonia solani and showed that its antifungal activity is, in part, attributed to the production of the chlorinated 9-amino-acid lipopeptide thanamycin (Mendes et al., 2011). To get more insight into its biosynthetic repertoire, the genome of Pseudomonas sp. SH-C52 was sequenced and subjected to in silico, mutational and functional analyses. The sequencing revealed a genome size of 6.3 Mb and 5579 predicted ORFs. Phylogenetic analysis placed strain SH-C52 within the Pseudomonas corrugata clade. In silico analysis for secondary metabolites revealed a total of six non-ribosomal peptide synthetase (NRPS) gene clusters, including the two previously described NRPS clusters for thanamycin and the 2-amino acid antibacterial lipopeptide brabantamide. Here we show that thanamycin also has activity against an array of other fungi and that brabantamide A exhibits anti-oomycete activity and affects phospholipases of the late blight pathogen Phytophthora infestans. Most notably, mass spectrometry led to the discovery of a third lipopeptide, designated thanapeptin, with a 22-amino-acid peptide moiety. Seven structural variants of thanapeptin were found with varying degrees of activity against P. infestans. Of the remaining four NRPS clusters, one was predicted to encode for yet another and unknown lipopeptide with a predicted peptide moiety of 8-amino acids. Collectively, these results show an enormous metabolic potential for Pseudomonas sp. SH-C52, with at least three structurally diverse lipopeptides, each with a different antimicrobial activity spectrum.
Immune activation mediated by the late blight resistance protein R1 requires nuclear localization of R1 and AVR1
Du, Y. ; Berg, J. ; Govers, F. ; Bouwmeester, K. - \ 2015
New Phytologist 207 (2015)3. - ISSN 0028-646X - p. 735 - 747.
disease-resistance - phytophthora-infestans - arabidopsis-thaliana - innate immunity - plant immunity - receptor - recognition - potato - gene - component
Resistance against oomycete pathogens is mainly governed by intracellular nucleotide-binding leucine-rich repeat (NLR) receptors that recognize matching avirulence (AVR) proteins from the pathogen, RXLR effectors that are delivered inside host cells. Detailed molecular understanding of how and where NLR proteins and RXLR effectors interact is essential to inform the deployment of durable resistance (R) genes. Fluorescent tags, nuclear localization signals (NLSs) and nuclear export signals (NESs) were exploited to determine the subcellular localization of the potato late blight protein R1 and the Phytophthora infestans RXLR effector AVR1, and to target these proteins to the nucleus or cytoplasm. Microscopic imaging revealed that both R1 and AVR1 occurred in the nucleus and cytoplasm, and were in close proximity. Transient expression of NLS- or NES-tagged R1 and AVR1 in Nicotiana benthamiana showed that activation of the R1-mediated hypersensitive response and resistance required localization of the R1/AVR1 pair in the nucleus. However, AVR1-mediated suppression of cell death in the absence of R1 was dependent on localization of AVR1 in the cytoplasm. Balanced nucleocytoplasmic partitioning of AVR1 seems to be a prerequisite. Our results show that R1-mediated immunity is activated inside the nucleus with AVR1 in close proximity and suggest that nucleocytoplasmic transport of R1 and AVR1 is tightly regulated.
Characterisation of the late blight resistance in potato differential MaR9 reveals a qualitative resistance gene, R9a, residing in a cluster of Tm-22 homologs on chromosome IX
Jo, K.R. ; Visser, R.G.F. ; Jacobsen, E. ; Vossen, J.H. - \ 2015
Theoretical and Applied Genetics 128 (2015)5. - ISSN 0040-5752 - p. 931 - 941.
allele conferring resistance - broad-spectrum resistance - race-specific resistance - phytophthora-infestans - disease resistance - r-gene - solanum-tuberosum - hypersensitive resistance - field-resistance - famine fungus
Late blight of potato (Solanum tuberosum), caused by Phytophthora infestans, can effectively be managed by genetic resistance. The MaR9 differential plant provides durable resistance to a broad spectrum of late blight strains. This resistance is brought about by at least seven genes derived from S. demissum including R1, Rpiabpt1, R3a, R3b, R4, R8 and, so far uncharacterized resistance gene(s). Here we set out to genetically characterize this additional resistance in MaR9. Three BC1 populations derived from MaR9 were identified that segregated for IPO-C resistance but that lacked R8. One BC1 population IX PCR marker, 184-81, fully co-segregated with R9a. The map position of R9a on the distal end of the lower arm of chromosome IX was confirmed using PCR markers GP101 and Stm1021. Successively, cluster-directed profiling (CDP) was carried out, revealing six closely linked markers. CDPSw58, CDPSw59 and CDPSw510 flanked the R9a gene at the distal end (5.8 cM) and, as expected, were highly homologous to Sw-5. CDPTm22 flanked R9a on the proximal side (2.9 cM). CDPTm26 and CDPTm27 fully cosegregated with resistance and had high homology to Tm-22, showing that R9a resides in a cluster of NBS–LRR genes with homology to Tm-22. Besides R9a, additional resistance of quantitative nature is found in MaR9, which remains to be genetically characterized. showed a continuous scale of resistance phenotypes, suggesting that multiple quantitative resistance genes were segregating. In two other BC1 populations esistance and susceptibility were segregating in a 1:1 ratio, suggesting a single qualitative resistance gene (R9a). A chromosome IX PCR marker, 184-81, fully co-segregated with R9a. The map position of R9a on the distal end of the lower arm of chromosome IX was confirmed using PCR markers GP101 and Stm1021. Successively, cluster-directed profiling (CDP) was carried out, revealing six closely linked markers. CDPSw58, CDPSw59 and CDPSw510 flanked the R9a gene at the distal end (5.8 cM) and, as expected, were highly homologous to Sw-5. CDPTm22 flanked R9a on the proximal side (2.9 cM). CDPTm26 and CDPTm27 fully cosegregated with resistance and had high homology to Tm-22, showing that R9a resides in a cluster of NBS–LRR genes with homology to Tm-22. Besides R9a, additional resistance of quantitative nature is found in MaR9, which remains to be genetically characterized.
Ex Situ Conservation Priorities for the Wild Relatives of Potato (Solanum L. Section Petota)
Castaneda-Alvarez, N.P. ; Haan, S. de; Juarez, H. ; Khoury, C.K. ; Achicanoy, H.A. ; Sosa, C.C. ; Bernau, V. ; Salas, A. ; Heider, B. ; Simon, R. ; Maxted, N. ; Spooner, D.M. - \ 2015
PLoS ONE 10 (2015)4. - ISSN 1932-6203 - 19 p.
globodera-pallida stone - late blight resistance - tuber-bearing solanum - somatic hybrids - cultivated potato - phytophthora-infestans - ralstonia-solanacearum - species distributions - ortholog sequences - genetic-resources
Crop wild relatives have a long history of use in potato breeding, particularly for pest and disease resistance, and are expected to be increasingly used in the search for tolerance to biotic and abiotic stresses. Their current and future use in crop improvement depends on their availability in ex situ germplasm collections. As these plants are impacted in the wild by habitat destruction and climate change, actions to ensure their conservation ex situ become ever more urgent. We analyzed the state of ex situ conservation of 73 of the closest wild relatives of potato (Solanum section Petota) with the aim of establishing priorities for further collecting to fill important gaps in germplasm collections. A total of 32 species (43.8%), were assigned high priority for further collecting due to severe gaps in their ex situ collections. Such gaps are most pronounced in the geographic center of diversity of the wild relatives in Peru. A total of 20 and 18 species were assessed as medium and low priority for further collecting, respectively, with only three species determined to be sufficiently represented currently. Priorities for further collecting include: (i) species completely lacking representation in germplasm collections; (ii) other high priority taxa, with geographic emphasis on the center of species diversity; (iii) medium priority species. Such collecting efforts combined with further emphasis on improving ex situ conservation technologies and methods, performing genotypic and phenotypic characterization of wild relative diversity, monitoring wild populations in situ, and making conserved wild relatives and their associated data accessible to the global research community, represent key steps in ensuring the long-term availability of the wild genetic resources of this important crop.
The Rsm regulon of plant growth-promoting Pseudomonas fluorescens SS101: role of small RNAs in regulation of lipopeptide biosynthesis
Song, C. ; Voort, M. van der; Mortel, J. van de; Hassan, K.A. ; Elbourne, L.D.H. ; Paulsen, I.T. ; Loper, J.E. ; Raaijmakers, J.M. - \ 2015
Microbial Biotechnology 8 (2015)2. - ISSN 1751-7907 - p. 296 - 310.
signal-transduction pathway - escherichia-coli - phytophthora-infestans - bacterial genomes - soluble-rnas - fumarase c - aeruginosa - identification - cha0 - recognition
The rhizobacterium Pseudomonas fluorescens SS101 inhibits growth of oomycete and fungal pathogens, and induces resistance in plants against pathogens and insects. To unravel regulatory pathways of secondary metabolite production in SS101, we conducted a genome-wide search for sRNAs and performed transcriptomic analyses to identify genes associated with the Rsm (repressor of secondary metabolites) regulon. In silico analysis led to the identification of 16 putative sRNAs in the SS101 genome. In frame deletion of the sRNAs rsmY and rsmZ showed that the Rsm system regulates the biosynthesis of the lipopeptide massetolide A and involves the two repressor proteins RsmA and RsmE, with the LuxR-type transcriptional regulator MassAR as their most likely target. Transcriptome analyses of the rsmYZ mutant further revealed that genes associated with iron acquisition, motility and chemotaxis were significantly upregulated, whereas genes of the type VI secretion system were downregulated. Comparative transcriptomic analyses showed that most, but not all, of the genes controlled by RsmY/RsmZ are also controlled by the GacS/GacA two-component system. We conclude that the Rsm regulon of P.¿fluorescens SS101 plays a critical role in the regulation of lipopeptide biosynthesis and controls the expression of other genes involved in motility, competition and survival in the plant rhizosphere.
The Top 10 oomycete pathogens in molecular plant pathology
Kamoun, S. ; Furzer, O. ; Jones, J.D.G. ; Judelson, H.S. ; Ali, G.S. ; Dalio, R.J.D. ; Roy, S.G. ; Schena, L. ; Zambounis, A. ; Panabières, F. ; Cahill, D. ; Ruocco, M. ; Figueiredo, A. ; Chen, X.R. ; Hulvey, J. ; Stam, R. ; Lamour, K. ; Gijzen, M. ; Tyler, B.M. ; Grünwald, N.J. ; Mukhtar, M.S. ; Tomé, D.F.A. ; Tör, M. ; Ackerveken, G. van den; McDowell, J. ; Daayf, F. ; Fry, W.E. ; Lindqvist-Kreuze, H. ; Meijer, H.J.G. ; Petre, B. ; Ristaino, J. ; Yoshida, K. ; Birch, P.R.J. ; Govers, F. - \ 2015
Molecular Plant Pathology 16 (2015)4. - ISSN 1464-6722 - p. 413 - 434.
grapevine downy mildew - irish potato famine - sudden oak death - blister rusts albuginaceae - eastern united-states - 3 clonal lineages - nb-lrr gene - phytophthora-infestans - plasmopara-viticola - arabidopsis-thaliana
Oomycetes form a deep lineage of eukaryotic organisms that includes a large number of plant pathogens that threaten natural and managed ecosystems. We undertook a survey to query the community for their ranking of plant pathogenic oomycete species based on scientific and economic importance. In total, we received 263 votes from 62 scientists in 15 countries for a total of 33 species. The Top 10 species and their ranking are: (1) Phytophthora infestans; (2, tied) Hyaloperonospora arabidopsidis; (2, tied) Phytophthora ramorum; (4) Phytophthora sojae; (5) Phytophthora capsici; (6) Plasmopara viticola; (7) Phytophthora cinnamomi; (8, tied) Phytophthora parasitica; (8, tied) Pythium ultimum; and (10) Albugo candida. The article provides an introduction to these 10 taxa and a snapshot of current research. We hope that the list will serve as a benchmark for future trends in oomycete research.
Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana
Du, J. ; Rietman, H. ; Vleeshouwers, V.G.A.A. - \ 2014
Journal of Visualized Experiments (2014)83. - ISSN 1940-087X - 7 p.
late blight resistance - mediated plant transformation - phytophthora-infestans - pathogen phytophthora - disease resistance - effector proteins - gene-expression - binary vector - agrobacterium - recognition
Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other's results.
PIRIN2 stabilizes cysteine protease XCP2 and increases susceptibility to the vascular pathogen Ralstonia solanacearum in Arabidopsis
Zhang, B. ; Tremousaygue, D. ; Denancé, N. ; Esse, H.P. van; Hörger, A.C. ; Dabos, P. ; Goffner, D. ; Thomma, B.P.H.J. ; Hoorn, R.A.L. van der; Tuominen, H. - \ 2014
The Plant Journal 79 (2014)6. - ISSN 0960-7412 - p. 1009 - 1019.
programmed cell-death - nf-kappa-b - disease resistance - phytophthora-infestans - gene-expression - plants - xylem - thaliana - effector - defense
PIRIN (PRN) is a member of the functionally diverse cupin protein superfamily. There are four members of the Arabidopsis thaliana PRN family, but the roles of these proteins are largely unknown. Here we describe a function of the Arabidopsis PIRIN2 (PRN2) that is related to susceptibility to the bacterial plant pathogen Ralstonia solanacearum. Two prn2 mutant alleles displayed decreased disease development and bacterial growth in response to R. solanacearum infection. We elucidated the underlying molecular mechanism by analyzing PRN2 interactions with the papain-like cysteine proteases (PLCPs) XCP2, RD21A, and RD21B, all of which bound to PRN2 in yeast two-hybrid assays and in Arabidopsis protoplast co-immunoprecipitation assays. We show that XCP2 is stabilized by PRN2 through inhibition of its autolysis on the basis of PLCP activity profiling assays and enzymatic assays with recombinant protein. The stabilization of XCP2 by PRN2 was also confirmed in planta. Like prn2 mutants, an xcp2 single knockout mutant and xcp2 prn2 double knockout mutant displayed decreased susceptibility to R. solanacearum, suggesting that stabilization of XCP2 by PRN2 underlies susceptibility to R. solanacearum in Arabidopsis.
Phenotypic analyses of Arabidopsis T-DNA insertion lines and expression profiling reveal that multiple L-type lectin receptor kinases are involved in plant immunity
Wang, Y. ; Bouwmeester, K. ; Beseh, P. ; Shan, W. ; Govers, F. - \ 2014
Molecular Plant-Microbe Interactions 27 (2014)12. - ISSN 0894-0282 - p. 1390 - 1402.
pattern-triggered immunity - phytophthora-infestans - salicylic-acid - defense responses - innate immunity - thaliana - gene - resistance - biology - roles
L-type lectin receptor kinases (LecRKs) are membrane-spanning receptor-like kinases with putative roles in biotic and abiotic stress responses and in plant development. In Arabidopsis, 45 LecRKs were identified but their functions are largely unknown. Here, a systematic functional analysis was carried out by evaluating phenotypic changes of Arabidopsis LecRK T-DNA insertion lines in plant development and upon exposure to various external stimuli. None of the LecRK T-DNA insertion lines showed clear developmental changes, neither under normal conditions nor upon abiotic stress treatment. However, many of the T-DNA insertion lines showed altered resistance to Phytophthora brassicae, Phytophthora capsici, Pseudomonas syringae or Alternaria brassicicola. One mutant defective in LecRK-V.5 expression, was compromised in resistance to two Phytophthora spp. but showed enhanced resistance to P. syringae. LecRK-V.5 overexpression confirmed its dual role in resistance and susceptibility depending on the pathogen. Combined analysis of these phenotypic data and LecRK expression profiles retrieved from public datasets revealed that LecRKs which are hardly induced upon infection or even suppressed are also involved in pathogen resistance. Computed co-expression analysis revealed that LecRKs with similar function displayed diverse expression patterns. Since LecRKs are widespread in plants, the results presented here provide invaluable information for exploring the potential of LecRKs as novel sources of resistance in crops.
Genomic characterisation of the effector complement of the potato cyst nematode Globodera pallida
Thorpe, P. ; Mantelin, S. ; Cock, P.J.A. ; Blok, V.C. ; Coke, M.C. ; Evers-van den Akker, S. ; Guzeeva, E. ; Lilley, C.J. ; Smant, G. ; Reid, A.J. ; Wright, K.M. ; Urwin, P.E. ; Jones, J.T. - \ 2014
BMC Genomics 15 (2014). - ISSN 1471-2164 - 15 p.
plant-parasitic nematodes - esophageal gland-cells - heterodera-glycines - meloidogyne-incognita - phytophthora-infestans - arabidopsis-thaliana - chorismate mutase - giant-cells - protein - sequence
Background The potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure – the syncytium – which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium. Results The recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure. Conclusion This is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process.
Erosion of quantitative host resistance in the apple × Venturia inaequalis pathosystem
Caffier, V. ; Lasserre-Zuber, P. ; Giraud, M. ; Lascostes, M. ; Sievenard, R. ; LeMarquand, A. ; Weg, W.E. van de; Expert, P. ; Denancé, C. - \ 2014
Infection, Genetics and Evolution 27 (2014). - ISSN 1567-1348 - p. 481 - 489.
f-sp tritici - latent period - trait loci - disease resistance - broad-spectrum - phytophthora-infestans - differential selection - pathogen population - puccinia-triticina - erysiphe-graminis
Theoretical approaches predict that host quantitative resistance selects for pathogens with a high level of pathogenicity, leading to erosion of the resistance. This process of erosion has, however, rarely been experimentally demonstrated. To investigate the erosion of apple quantitative resistance to scab disease, we surveyed scab incidence over time in a network of three orchards planted with susceptible and quantitatively resistant apple genotypes. We sampled Venturiainaequalis isolates from two of these orchards at the beginning of the experiment and we tested their quantitative components of pathogenicity (i.e., global disease severity, lesion density, lesion size, latent period) under controlled conditions. The disease severity produced by the isolates on the quantitatively resistant apple genotypes differed between the sites. Our study showed that quantitative resistance may be subject to erosion and even complete breakdown, depending on the site. We observed this evolution over time for apple genotypes that combine two broad-spectrum scab resistance QTLs, F11 and F17, showing a significant synergic effect of this combination in favour of resistance (i.e., favourable epistatic effect). We showed that isolates sampled in the orchard where the resistance was inefficient presented a similar level of pathogenicity on both apple genotypes with quantitative resistance and susceptible genotypes. As a consequence, our results revealed a case where the use of quantitative resistance may result in the emergence of a generalist pathogen population that has extended its pathogenicity range by performing similarly on susceptible and resistant genotypes. This emphasizes the need to develop quantitative resistances conducive to trade-offs within the pathogen populations concerned.
Development of late blight resistant potatoes by cisgenic stacking
Jo, K.R. ; Kim, C.J. ; Kim, S.J. ; Kim, T.J. ; Bergervoet-van Deelen, J.E.M. ; Jongsma, M.A. ; Visser, R.G.F. ; Jacobsen, E. ; Vossen, J.H. - \ 2014
BMC Biotechnology 14 (2014). - ISSN 1472-6750
broad-spectrum resistance - cultivar sarpo mira - phytophthora-infestans - solanum-bulbocastanum - r-gene - plants - transformation - genomics - tomato - biotechnology
Background Phytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance (R) genes with different spectra from crossable species into potato varieties is required. Cisgenesis is a promising approach that introduces native genes from the crops own gene pool using GM technology, thereby retaining favourable characteristics of established varieties. Results We pursued a cisgenesis approach to introduce two broad spectrum potato late blight R genes, Rpi-sto1 and Rpi-vnt1.1 from the crossable species Solanum stoloniferum and Solanum venturii, respectively, into three different potato varieties. First, single R gene-containing transgenic plants were produced for all varieties to be used as references for the resistance levels and spectra to be expected in the respective genetic backgrounds. Next, a construct containing both cisgenic late blight R genes (Rpi-vnt1.1 and Rpi-sto1), but lacking the bacterial kanamycin resistance selection marker (NPTII) was transformed to the three selected potato varieties using Agrobacterium-mediated transformation. Gene transfer events were selected by PCR among regenerated shoots. Through further analyses involving morphological evaluations in the greenhouse, responsiveness to Avr genes and late blight resistance in detached leaf assays, the selection was narrowed down to eight independent events. These cisgenic events were selected because they showed broad spectrum late blight resistance due to the activity of both introduced R genes. The marker-free transformation was compared to kanamycin resistance assisted transformation in terms of T-DNA and vector backbone integration frequency. Also, differences in regeneration time and genotype dependency were evaluated. Conclusions We developed a marker-free transformation pipeline to select potato plants functionally expressing a stack of late blight R genes. Marker-free transformation is less genotype dependent and less prone to vector backbone integration as compared to marker-assisted transformation. Thereby, this study provides an important tool for the successful deployment of R genes in agriculture and contributes to the production of potentially durable late blight resistant potatoes.
Novel applications of motif-directed profiling to identify disease resistance genes in plants
Vossen, J.H. ; Dezhsetan, S. ; Esselink, D. ; Arens, M. ; Sanz, M.J. ; Verweij, W. ; Verzaux, E. ; Linden, C.G. van der - \ 2013
Plant Methods 9 (2013). - ISSN 1746-4811
nucleotide-binding-site - kinase-like protein - phytophthora-infestans - conferring resistance - fusarium-oxysporum - diploid avena - potato - map - loci - encodes
Background: Molecular profiling of gene families is a versatile tool to study diversity between individual genomes in sexual crosses and germplasm. Nucleotide binding site (NBS) profiling, in particular, targets conserved nucleotide binding site-encoding sequences of resistance gene analogs (RGAs), and is widely used to identify molecular markers for disease resistance (R) genes. Results: In this study, we used NBS profiling to identify genome-wide locations of RGA clusters in the genome of potato clone RH. Positions of RGAs in the potato RH and DM genomes that were generated using profiling and genome sequencing, respectively, were compared. Largely overlapping results, but also interesting discrepancies, were found. Due to the clustering of RGAs, several parts of the genome are overexposed while others remain underexposed using NBS profiling. It is shown how the profiling of other gene families, i.e. protein kinases and different protein domain-coding sequences (i.e., TIR), can be used to achieve a better marker distribution. The power of profiling techniques is further illustrated using RGA cluster-directed profiling in a population of Solanum berthaultii. Multiple different paralogous RGAs within the Rpi-ber cluster could be genetically distinguished. Finally, an adaptation of the profiling protocol was made that allowed the parallel sequencing of profiling fragments using next generation sequencing. The types of RGAs that were tagged in this next-generation profiling approach largely overlapped with classical gel-based profiling. As a potential application of next-generation profiling, we showed how the R gene family associated with late blight resistance in the SH*RH population could be identified using a bulked segregant approach. Conclusions: In this study, we provide a comprehensive overview of previously described and novel profiling primers and their genomic targets in potato through genetic mapping and comparative genomics. Furthermore, it is shown how genome-wide or fine mapping can be pursued by choosing different sets of profiling primers. A protocol for next-generation profiling is provided and will form the basis for novel applications. Using the current overview of genomic targets, a rational choice can be made for profiling primers to be employed.
Genomic analysis of the native European Solanum species, S. dulcamara
Agostino, N.D. ; Golas, T. ; Geest, H. van; Bombarely, A. ; Dawood, T. ; Zethof, J. ; Driedonks, N. ; Wijnker, T.G. ; Bargsten, J. ; Nap, J.P. ; Mariani, C. ; Rieu, I. - \ 2013
BMC Genomics 14 (2013). - ISSN 1471-2164 - 14 p.
phytophthora-infestans - solanaceae - identification - evolution - polymorphism - sequence - tomato - potato - genes
Background - Solanum dulcamara (bittersweet, climbing nightshade) is one of the few species of the Solanaceae family native to Europe. As a common weed it is adapted to a wide range of ecological niches and it has long been recognized as one of the alternative hosts for pathogens and pests responsible for many important diseases in potato, such as Phytophthora. At the same time, it may represent an alternative source of resistance genes against these diseases. Despite its unique ecology and potential as a genetic resource, genomic research tools are lacking for S. dulcamara. We have taken advantage of next-generation sequencing to speed up research on and use of this non-model species. Results - In this work, we present the first large-scale characterization of the S. dulcamara transcriptome. Through comparison of RNAseq reads from two different accessions, we were able to predict transcript-based SNP and SSR markers. Using the SNP markers in combination with genomic AFLP and CAPS markers, the first genome-wide genetic linkage map of bittersweet was generated. Based on gene orthology, the markers were anchored to the genome of related Solanum species (tomato, potato and eggplant), revealing both conserved and novel chromosomal rearrangements. This allowed a better estimation of the evolutionary moment of rearrangements in a number of cases and showed that chromosomal breakpoints are regularly re-used. Conclusion - Knowledge and tools developed as part of this study pave the way for future genomic research and exploitation of this wild Solanum species. The transcriptome assembly represents a resource for functional analysis of genes underlying interesting biological and agronomical traits and, in the absence of the full genome, provides a reference for RNAseq gene expression profiling aimed at understanding the unique biology of S. dulcamara. Cross-species orthology-based marker selection is shown to be a powerful tool to quickly generate a comparative genetic map, which may speed up gene mapping and contribute to the understanding of genome evolution within the Solanaceae family.
Specific In Planta Recognition of Two GKLR Proteins of the Downy Mildew Bremia lactucae Revealed in a Large Effector Screen in Lettuce
Stassen, J.H.M. ; Boer, E. den; Vergeer, P.W.J. ; Andel, A. ; Ellendorff, U. ; Pelgrom, K.T.B. ; Pel, M. ; Schut, J. ; Zonneveld, O. ; Jeuken, M.J.W. ; Ackerveken, G. van den - \ 2013
Molecular Plant-Microbe Interactions 26 (2013)11. - ISSN 0894-0282 - p. 1259 - 1270.
backcross inbred lines - genetic-linkage map - disease resistance - phytophthora-infestans - nonhost resistance - avirulence genes - wild lettuce - pathogen - saligna - potato
Breeding lettuce (Lactuca sativa) for resistance to the downy mildew pathogen Bremia lactucae is mainly achieved by introgression of dominant downy mildew resistance (Dm) genes. New Bremia races quickly render Dm genes ineffective, possibly by mutation of recognized host-translocated effectors or by suppression of effector-triggered immunity. We have previously identified 34 potential RXLR(-like) effector proteins of B. lactucae that were here tested for specific recognition within a collection of 129 B. lactucae-resistant Lactuca lines. Two effectors triggered a hypersensitive response: BLG01 in 52 lines, predominantly L. saligna, and BLG03 in two L. sativa lines containing Dm2 resistance. The N-terminal sequences of BLG01 and BLG03, containing the signal peptide and GKLR variant of the RXLR translocation motif, are not required for in planta recognition but function in effector delivery. The locus responsible for BLG01 recognition maps to the bottom of lettuce chromosome 9, whereas recognition of BLG03 maps in the RGC2 cluster on chromosome 2. Lactuca lines that recognize the BLG effectors are not resistant to Bremia isolate Bl:24 that expresses both BLG genes, suggesting that Bl:24 can suppress the triggered immune responses. In contrast, lettuce segregants displaying Dm2-mediated resistance to Bremia isolate Bl:5 are responsive to BLG03, suggesting that BLG03 is a candidate Avr2 protein.
Climate Change and Potato Production in Contrasting South African Agro-Ecosystems 3. Effects on Relative Development Rates of Selected Pathogens and Pests
Waals, J.E. van der; Krüger, K. ; Franke, A.C. ; Haverkort, A.J. ; Steyn, J.M. - \ 2013
Potato Research 56 (2013)1. - ISSN 0014-3065 - p. 67 - 84.
late-blight epidemics - phytophthora-infestans - meloidogyne-incognita - myzus-persicae - virus-y - transmission efficiency - alternaria-solani - plant-diseases - soil texture - elevated co2
A set of daily weather data simulations for 1961 to 2050 were used to calculate past and future trends in pest and disease pressure in potato cropping systems at three agro-ecologically distinct sites in South Africa: the Sandveld, the Eastern Free State and Limpopo. The diseases and pests modelled were late blight, early blight and brown spot, blackleg and soft rot, root-knot nematodes and the peach-potato aphid Myzus persicae (as indicator of Potato virus Y and Potato leaf roll virus). The effects of climate on trends in relative development rates of these pathogens and pests were modelled for each pathogen and pest using a set of quantitative parameters, which included specific temperature and moisture requirements for population growth, compiled from literature. Results showed that the cumulative relative development rate (cRDR) of soft rot and blackleg, root-knot nematodes and M. persicae will increase over the 90-year period in the areas under consideration. The cRDR of early blight and brown spot is likely to increase in the wet winter and wet summer crops of the Sandveld and Eastern Free State, respectively, but remains unchanged in the dry summer and dry winter crops of the Sandveld and Limpopo, respectively. Climate change will decrease the cRDR of late blight in all of the cropping systems modelled, except in the wet winter crop of the Sandveld. These results help to set priorities in research and breeding, specifically in relation to management strategies for diseases and pests.
Phenotypic, Molecular, and Pathological Characterization of Colletotrichum acutatum Associated with Andean Lupine and Tamarillo in the Ecuadorian Andes
Falconi, C. ; Visser, R.G.F. ; Heusden, A.W. van - \ 2013
Plant Disease 97 (2013)6. - ISSN 0191-2917 - p. 819 - 827.
phytophthora-infestans - phylogenetic-relationships - olive anthracnose - ribosomal dna - sensu-lato - strawberry - identification - diversity - gloeosporioides - tomato
Anthracnose is a serious problem of both Andean lupine and tamarillo in Ecuador. Morphological features, internal transcribed spacer (ITS) sequences, and host specificity were used to characterize Colletotrichum isolates from lupine and tamarillo. Based on phenotypic and molecular characterization, the causal agent of anthracnose on both hosts was Colletotrichum acutatum. All isolates were identified in a C. acutatum-specific polymerase chain reaction assay. Colony diameter, conidia shape, and insensitivity to benomyl also placed isolates from both hosts in the C. acutatum group. However, a detailed analysis of the ITS sequences placed the lupine and tamarillo isolates from the Ecuadorian Andean zone in two clades, with both lupine and tamarillo isolates in each clade. C. acutatum isolates from Andean lupine were distinct from other C. acutatum isolates on lupine around the world. In cross-infection studies, the diameter of lesions produced by isolates from each host was compared on the main stem of two tamarillo and three lupine cultivars. Some isolates produced larger lesions on the host from which they were isolated but others showed similar aggressiveness on their alternate host. Isolates from both hosts were biotrophic on lupine stems, producing little necrosis and abundant sporulation whereas, on tamarillo stems, they produced dark lesions with few conidia. The collection of C. acutatum isolates from lupine and tamarillo provides interesting material for the study quantitative host adaptation.
Vector integration in triple R gene transformants and the clustered inheritance of resistance against potato late blight
Zhu, S. ; Duwal, A. ; Su, Q. ; Vossen, J.H. ; Visser, R.G.F. ; Jacobsen, E. - \ 2013
Transgenic Research 22 (2013)2. - ISSN 0962-8819 - p. 315 - 325.
agrobacterium-mediated transformation - phytophthora-infestans - backbone sequences - transgene expression - plant transformation - dna transfer - genome - rice - tumefaciens - chromosome
Genetic transformation with resistance (R) genes is expected to enhance resistance durability against pathogens, especially for potato, a vegetatively propagated crop with tetrasomic inheritance and a long-term breeding program. In this study, 128 potato transformants were analysed for the presence of vector T-DNA genes, borders and backbone sequences. They were harvested after transformation using a construct containing neomycin phosphotransferase II (nptII) and three R genes against potato late blight (Phytophthora infestans). Our analysis revealed that 45 % of the R gene-containing transformants possessed a low T-DNA copy number, without the integration of vector backbone and borders. The integration of vector backbone sequences was characterized using eight genes, and backbone gene tetA was selected for the early prediction of plants with backbone sequence integration. Three transformants, two plants harbouring one T-DNA copy and one plant harbouring three T-DNA copies, were crossed with susceptible cv. Katahdin. Based on our results, we conclude that all four T-DNA genes were inherited as one cluster and segregated in a Mendelian fashion. The three T-DNA inserts from the transformant harbouring three T-DNA copies were statistically proven to be un-linked and inherited into the offspring plants independently. All of the R genes were functionally expressed in the offspring plants as in their parental transformants. This functional gene stacking has important implications towards achieving more durable resistance against potato late blight
Reconstruction of Oomycete Genome Evolution Identifies Differences in Evolutionary Trajectories Leading to Present-Day Large Gene Families
Seidl, M.F. ; Ackerveken, G. van den; Govers, F. ; Snel, B. - \ 2012
Genome Biology and Evolution 4 (2012)3. - ISSN 1759-6653 - p. 199 - 211.
nonphotosynthetic protists - phytophthora-infestans - pathogen phytophthora - obligate biotrophy - protein families - plant-pathogens - duplication - reveals - cells - mechanisms
The taxonomic class of oomycetes contains numerous pathogens of plants and animals but is related to nonpathogenic diatoms and brown algae. Oomycetes have flexible genomes comprising large gene families that play roles in pathogenicity. The evolutionary processes that shaped the gene content have not yet been studied by applying systematic tree reconciliation of the phylome of these species. We analyzed evolutionary dynamics of ten Stramenopiles. Gene gains, duplications, and losses were inferred by tree reconciliation of 18,459 gene trees constituting the phylome with a highly supported species phylogeny. We reconstructed a strikingly large last common ancestor of the Stramenopiles that contained ~10,000 genes. Throughout evolution, the genomes of pathogenic oomycetes have constantly gained and lost genes, though gene gains through duplications outnumber the losses. The branch leading to the plant pathogenic Phytophthora genus was identified as a major transition point characterized by increased frequency of duplication events that has likely driven the speciation within this genus. Large gene families encoding different classes of enzymes associated with pathogenicity such as glycoside hydrolases are formed by complex and distinct patterns of duplications and losses leading to their expansion in extant oomycetes. This study unveils the large-scale evolutionary dynamics that shaped the genomes of pathogenic oomycetes. By the application of phylogenetic based analyses methods, it provides additional insights that shed light on the complex history of oomycete genome evolution and the emergence of large gene families characteristic for this important class of pathogens