Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Virulence contribution and recognition of homologs of the Verticillium dahliae effector Ave1
Boshoven, Jordi C. - \ 2017
University. Promotor(en): Bart Thomma; Pierre de Wit. - Wageningen : Wageningen University - ISBN 9789463436441 - 183
verticillium dahliae - plant pathogenic fungi - plant pathogens - disease resistance - virulence factors - virulence - immunity - host parasite relationships - plant-microbe interactions - symbiosis - mutagenesis - resistance breeding - plantenziekteverwekkende schimmels - plantenziekteverwekkers - ziekteresistentie - virulente factoren - virulentie - immuniteit - gastheer parasiet relaties - plant-microbe interacties - symbiose - mutagenese - resistentieveredeling

Disease resistance in crops is an important aspect of securing global food security. Resistant plants carry immune receptors that sense pathogen invasion often through the recognition of important pathogen virulence factors, known as effectors. Thus, identification and characterization of effectors is important for the fundamental understanding of virulence mechanisms and to aid in resistance breeding. In this thesis the VdAve1 effector of the soil-borne fungal pathogen Verticillium dahliae is studied that is recognized by tomato immune receptor Ve1. Homologs were found in other plant pathogens and the role in virulence in these pathogens was analyzed. Ave1 homologs are differentially recognized by Ve1 and with a combination of domain swaps and truncations a surface exposed patch was identified that contributes to the recognition by Ve1. Knowledge of specific effector-receptor combinations and knowledge of effectors in general can be exploited to aid in breeding for durable resistance in crops.

Recognition of Verticillium effector Ave1 by tomato immune receptor Ve1 mediates Verticillium resistance in diverse plant species
Song, Yin - \ 2017
University. Promotor(en): Bart Thomma; Pierre de Wit. - Wageningen : Wageningen University - ISBN 9789463437950 - 231
disease resistance - defence mechanisms - immunity - plant-microbe interactions - plant pathogens - verticillium dahliae - verticillium - tomatoes - solanum lycopersicum - receptors - genes - tobacco - nicotiana glutinosa - potatoes - solanum tuberosum - solanum torvum - humulus lupulus - cotton - gossypium hirsutum - transgenic plants - arabidopsis thaliana - ziekteresistentie - verdedigingsmechanismen - immuniteit - plant-microbe interacties - plantenziekteverwekkers - tomaten - receptoren - genen - tabak - aardappelen - katoen - transgene planten

Plant-pathogenic microbes secrete effector molecules to establish disease on their hosts, whereas plants in turn employ immune receptors to try and intercept such effectors in order to prevent pathogen colonization. Based on structure and subcellular location, immune receptors fall into two major classes; cell surface-localized receptors that comprise receptor kinases (RKs) and receptor-like proteins (RLPs) that monitor the extracellular space, and cytoplasm-localized nucleotide-binding domain leucine-rich repeat receptors (NLRs) that survey the intracellular environment. Race-specific resistance to Verticillium wilt in tomato (Solanum lycopersicum) is governed by the tomato extracellular leucine-rich repeat (eLRR)-containing RLP-type cell surface receptor Ve1 upon recognition of the effector protein Ave1 that is secreted by race 1 strains of the soil-borne vascular wilt Verticillium dahliae. Homologues of V. dahliae Ave1 (VdAve1) are found in plants and in a number of plant pathogenic microbes, and some of these VdAve1 homologues are recognized by tomato Ve1. The research presented in this thesis aims to characterize the role of the tomato cell surface-localized immune receptor Ve1, and its homologues in other diverse plant species, in Verticillium wilt resistance.

Pedigree-based analysis in a multiparental population of octoploid strawberry reveals QTL alleles conferring resistance to phytophthora cactorum
Mangandi, Jozer ; Verma, Sujeet ; Osorio, Luis ; Peres, Natalia A. ; Weg, Eric van de; Whitaker, Vance M. - \ 2017
G3 : Genes Genomes Genetics 7 (2017)6. - ISSN 2160-1836 - p. 1707 - 1719.
disease resistance - FaRPc2 - FlexQTL - Fragaria - Haplotype quantitative trait locus - MPP - Multiparental population

Understanding the genetic architecture of traits in breeding programs can be critical for making genetic progress. Important factors include the number of loci controlling a trait, allele frequencies at those loci, and allele effects in breeding germplasm. To this end, multiparental populations offer many advantages for quantitative trait locus (QTL) analyses compared to biparental populations. These include increased power for QTL detection, the ability to sample a larger number of segregating loci and alleles, and estimation of allele effects across diverse genetic backgrounds. Here, we investigate the genetic architecture of resistance to crown rot disease caused by Phytophthora cactorum in strawberry (Fragaria × ananassa), using connected full-sib families from a breeding population. Clonal replicates of > 1100 seedlings from 139 full-sib families arising from 61 parents were control-inoculated during two consecutive seasons. Subgenome-specific single nucleotide polymorphism (SNP) loci were mapped in allo-octoploid strawberry (2n = 8 × = 56), and FlexQTL software was utilized to perform a Bayesian, pedigree-based QTL analysis. A major locus on linkage group (LG) 7D, which we name FaRPc2, accounts for most of the genetic variation for resistance. Four predominant SNP haplotypes were detected in the FaRPc2 region, two of which are strongly associated with two different levels of resistance, suggesting the presence of multiple resistance alleles. The phenotypic effects of FaRPc2 alleles across trials and across numerous genetic backgrounds make this locus a highly desirable target for genetic improvement of resistance in cultivated strawberry.

Unraveling the genetics of Botrytis cinerea resistance in Gerbera hybrida
Fu, Yiqian - \ 2017
University. Promotor(en): Richard Visser, co-promotor(en): Paul Arens; Jaap van Tuyl. - Wageningen : Wageningen University - ISBN 9789463431811 - 159
gerbera - plant pathogenic fungi - botrytis cinerea - disease resistance - genetic mapping - transcriptomics - quantitative trait loci - plant breeding - plantenziekteverwekkende schimmels - ziekteresistentie - genetische kartering - transcriptomica - loci voor kwantitatief kenmerk - plantenveredeling

Gerbera hybrida is one of the top five cut flowers. It is well-known to people for its variation in flower color and patterning. Gerbera breeding at the moment is done using conventional methods which are based on a phenotypic selection. This has drawbacks in breeding speed and efficiency, especially for complex traits like disease resistance. Gerbera gray mold, promoted by high humidity during the production in greenhouses or by an accumulation of condensate during transportation, is a considerable threat to the gerbera production. Gerbera gray mold is caused by Botrytis cinerea and plant resistance to B. cinerea is considered to be a polygenic trait that needs the contribution of multiple loci, and on top of that is highly affected by the environment. Conventional breeding might be inefficient for improving Botrytis resistance in gerbera.

In this study, the transcriptomes of four parents of two gerbera populations were sequenced using Illumina paired-end sequencing. Transcriptome data provides a resource for genetic dissection and an insight to explore gene functions for this ornamental crop. To identify the QTL regions leading to the phenotypic variation in Botrytis resistance, and establishing a relationship between marker genotype and phenotypic variation for marker assisted selection (MAS), genetic linkage maps were constructed with SNP markers in the two F1 segregating populations. A total of 20 QTLs were identified in the parental maps of the two populations. The number of QTLs found and the explained variance of most QTLs detected reflects the complex mechanism of Botrytis disease response. Narrowing down the QTL region and identifying the causal gene(s) underlying a QTL could maximize the effective use of MAS in breeding. Homologs of known functional genes involved in Botrytis resistance from other species were obtained in gerbera and SNP markers identified and mapped. Twenty-nine candidate genes were mapped and seven candidate genes could be mapped on both populations. Seven candidate genes were located in the vicinity of the QTLs detected. The co-localization of QTLs with CGs gives an indication that these candidate genes could probably be involved in resistance to Botrytis and provide a more precise possibility to use MAS in gerbera breeding in the future. A tobacco rattle virus (TRV) based gene silencing system which was used to inspect the function of two candidate genes. The two CGs are the homologs of the genes responsible for Botrytis resistance in tomato and both mapped in QTL regions related to Botrytis resistance in gerbera ray floret test. Silencing the two genes by VIGS, showed smaller lesion sizes upon Botrytis infection on gerbera ray florets compared with the controls.

The entire research went from the generation of four parental transcriptome data sets to development of SNP markers (Chapter 2), construction of genetic maps and to mapping QTLs for Botrytis resistance (Chapter 3). This was further on combined with candidate gene searching in other crops, querying and mapping homologous genes (Chapter 4) and characterizing the candidate genes which co-localized with QTLs (Chapter 5). The whole process not only helped us to unravel the genetics of Botrytis resistance in gerbera and develop genetic tools for gerbera improvement, but also could serve as guidance for developing marker-assisted selection for other ornamental plants from the beginning.

Potatoes, pathogens and pests : effects of genetic modifi cation for plant resistance on non-target arthropods
Lazebnik, Jenny - \ 2017
University. Promotor(en): Joop van Loon; Marcel Dicke. - Wageningen : Wageningen University - ISBN 9789463431620 - 151
solanum tuberosum - potatoes - oomycetes - phytophthora infestans - genetic engineering - transgenic plants - disease resistance - risk assessment - nontarget organisms - arthropods - insect pests - herbivores - trophic levels - ecological risk assessment - greenhouse experiments - field experimentation - aardappelen - oömyceten - genetische modificatie - transgene planten - ziekteresistentie - risicoschatting - niet-doelorganismen - geleedpotigen - insectenplagen - herbivoren - trofische graden - ecologische risicoschatting - kasproeven - experimenteel veldonderzoek

Currently, fungicides are necessary to protect potato crops against late blight, Phytophthora infestans, one of the world’s most damaging crop pathogens. The introgression of plant resistance genes from wild potato species targeted specifically to the late blight pathogen into susceptible potato varieties may alleviate the environmental impact of chemical control. Genetically modified plants are subject to an environmental risk assessment, and this includes testing for risks to the non-target arthropod community associated with the crop. The thesis begins with a review about the main plant defense responses and their role in influencing sequential interactions between herbivores and plant pathogens. The experimental chapters each focus on different aspects of the interaction between potato plants (both resistant and susceptible), the target pathogen (P. infestans) and several non-target insects. With each chapter, the scope widens: from the molecular gene expression in potato leaves in response to sequential attacks, to field scale biodiversity analyses. At the molecular level, one of the main findings was that the genomic position of the Rpi-vnt1 insertion conferring resistance to P. infestans influenced potato gene expression measured in leaves, when interacting with the non-target insect pests Myzus persicae (Green peach aphid) and Leptinotarsa decemlineata (Colorado potato beetle). Insect performance differed between the resistant GM and susceptible non-GM comparator. In the following chapter, the differences in insect performance were tested across a range of conventionally bred cultivars varying in resistance to P. infestans. Differences in M. persicae performance between several cultivars greatly outweighed the differences previously detected between the GM and non-GM comparator. These results are crucial in shaping the way risk is assessed in the context of GM crops, and these results are supported in our experiments assessing effects on biodiversity with pitfall traps in the field. The third trophic level was also addressed by comparing the performance of the parasitoid Aphidius colemani reared on GM and non-GM fed aphids, both with an without exposure to P. infestans. Differences in parasitoid performance were only found on the susceptible cultivar when inoculated with P. infestans. In the last experimental chapter the risk assessment is taken to the field comparing pitfall trap catches over two years and in two countries. Different methods for statistical analysis of biodiversity data were compared to arrive at recommendations for such analysis in the framework of environmental risk assessments. Drawing on these lessons, the discussion ends with ideas for the development of protocols for environmental risk assessments in the light of expected scientific progress in agricultural biotechnology.

Susceptibility genes : an additional source for improved resistance
Sun, Kaile - \ 2017
University. Promotor(en): Richard Visser, co-promotor(en): Evert Jacobsen; Yuling Bai. - Wageningen : Wageningen University - ISBN 9789463431415 - 174
solanum tuberosum - potatoes - solanum lycopersicum - tomatoes - genes - susceptibility - plant pathogenic fungi - phytophthora infestans - disease resistance - plant breeding - aardappelen - tomaten - genen - vatbaarheid - plantenziekteverwekkende schimmels - ziekteresistentie - plantenveredeling

Potato is affected by several diseases. Although, resistance can be obtained by introgression of major resistance genes from wild species, this has rarely been durable. Hence, other sources of resistance are highly needed. New research with a focus on loss of function mutations has led to the identification of disease susceptibility (S) genes in plants. The research in this thesis was aimed at the identification and characterization of potato S genes involved in the interaction with Phytophthora infestans and Botrytis cinerea. We selected 11 Arabidopsis thaliana S genes and silenced their potato orthologs by RNAi in the potato cultivar Desiree. The silencing of six genes resulted in resistance to P. infestans. Moreover, silencing of StDND1 reduced the infection of B. cinerea. Microscopic analysis showed that spore attachment and/or germination of P. infestans and B. cinerea was hampered on the leaf surface of StDND1-silenced potato plants. On StDMR1- and StDMR6-silenced potato plants, hyphal growth of P. infestans was arrested by the hypersensitive response-like cell death. Our results demonstrate that impairment of plant S genes may open a new way for breeding potatoes with resistance to pathogens like P. infestans and B. cinerea.

Hevige phytophthora-uitbraak 2016 benadrukt noodzaak resistente rassen
Lammerts Van Bueren, E. ; Hutten, R.C.B. ; Engelen, C.J.M. - \ 2016
Aardappelwereld 2016 (2016)9. - ISSN 0169-653X - p. 32 - 33.
phytophthora infestans - aardappelen - biologische landbouw - ziekteresistentie - resistentie van variëteiten - rassen (planten) - plantenveredeling - potatoes - organic farming - disease resistance - varietal resistance - varieties - plant breeding
De biologische aardappelteelt heeft het in 2016 weer zwaar te verduren gehad met de hevige uitbraak van Phytophtora infestans. Door aanhoudende regenval in juni en juli waren veel telers genoodzaakt om hun percelen met aangetaste aardappelen al vroeg in het seizoen te branden. Gecombineerd met de late pootdatum hebben velen een (te) lage opbrengst van hun vatbare rassen dit jaar. "DIt bevestigt nog maar weer eens de noodzaak van resistente rassen", zo stellen Edith Lammerts van Bueren, Ronald Hutten en Christel Engelen, van het project Bioimpuls waar de deelnemers hard aan nieuwe resistente rassen werken.
Susceptibility pays off: insights into the mlo-based powdery mildew resistance
Appiano, Michela - \ 2016
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 - tomaten - ziekteresistentie - vatbaarheid - 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.

Biomarkers and mechanisms of natural disease resistance in dairy cows
Altena, S.E.C. van - \ 2016
University. Promotor(en): Huub Savelkoul, co-promotor(en): Edwin Tijhaar. - Wageningen : Wageningen University - ISBN 9789462578005 - 158 p.
dairy cows - biomarkers - disease resistance - immunity - antibodies - proteomics - immune response - dendritic cells - immunology - melkkoeien - ziekteresistentie - immuniteit - antilichamen - eiwitexpressieanalyse - immuniteitsreactie - dendritische cellen - immunologie

The aim of this thesis was to define and test biomarkers for disease resistance in dairy cows and to determine the underlying mechanism in natural disease resistance. The health status of the cows is an important issue in dairy farming. Due to the mandatory reduction in the use of antibiotics, alternatives are required to prevent the development and expression of illness in dairy cows. The identification of biomarkers associated with such disease offers the opportunity to adapt the management of cows at risk, and in this way, prevent them from developing overt disease. Previously, natural antibodies (NAbs) in serum and milk were used as candidate biomarkers for natural disease resistance in cows. In this thesis, we continue on the occurrence and mode of action of NAbs and also focus on their source: the B-1 cells. We performed a literature study on the identification and function of B-1 cells in different species and defined the limitations in the current identification of these cells in pigs, sheep and cows (Chapter 2). B-1 cells were described in cows by using widely accepted cell surface markers CD5 and CD11b. However, in literature several findings suggest that these cell surface markers are not unique markers for B-1 identification. The similarities between mice and veterinary animals in foetal B-cell development and antibody production, implies that B-1 cells are present in cows. In chapter 3, we carefully studied new markers to selectively identify B-1 cells in cows. The combination of B-1 cell markers IgM++ and pSYK++ (indicator constitutive intracellular signalling) identifies a distinct cell population with essential B-1 characteristics such as high CD80 expression. In addition, the development of these B-1 cells in calves before colostrum intake and 3 weeks afterwards shows the same kinetics as the development of NAbs represented by IgM antibodies binding to the well-accepted NAb-antigen phosphatidylcholine (PtC). In calves up to half a year of age, it is shown that the production of such NAbs increases from birth and stabilises from 6 weeks onwards. This implies an endogenous NAb production, which follows the same age-related kinetics as can be expected from B-1 cell development. In contrast, the development of total IgM antibody levels in calves shows a bimodal distribution, which is caused by the uptake and breakdown of maternally-derived IgM and simultaneous endogenous production of specific and natural IgM. Chapter 4 describes the role of such NAbs in bovine immunity. NAbs were represented by the binding of IgM to the naïve antigen keyhole limpet hemocyanin (KLH). Cows with high serum NAb levels were shown to have more IgM and IgG antibodies binding to common microbial structures LPS, LTA and PGN, than cows with low serum NAb levels. In addition, they also have more IgM antibodies binding to intact, fixed E. coli and S. Typhimurium bacteria. However, the killing of live E. coli and S. Typhimurium bacteria via antibody-mediated complement killing does not differ between cows with high and low NAb levels. The antibody-mediated complement killing was determined in a newly developed serum bactericidal test. Cows that performed less in the bactericidal test were more likely to develop mastitis in the future. This association was observed for the killing of E. coli and S. Typhimurium and the development of mastitis within the next one year. For S. Typhimurium the association was still present for the cases of mastitis occurring within four years after testing. Alternative biomarkers for disease resistance in cows were defined in chapter 5 by using a contemporary proteomics approach. Milk samples from high and low disease resistant cows were selected from the “Resilient Cattle” (Weerbaar Vee) biobank. Comparing the spectrum of milk proteins of high and low disease-resistant cows showed potential candidate biomarkers that were elevated in the milk of low-resistant cows. Two candidate marker proteins were validated with ELISA in a new and larger group of high- and low-resistant cows. Lactoferrin (LF) levels were significantly increased in milk of low-resistant cows. In addition, LF levels in milk were associated with clinical manifestations of lameness and had a predictive value for subsequent culling.

In conclusion, we found that also in cows NAbs are produced by B-1 cells that can be identified based on the combined expression of cell surface IgM and internal pSYK. In addition, the frequency of these B-1 cells after birth follows a similar kinetics as described before in mice. These NAbs can be more precisely identified based on their PtC binding ability and their functional activity in a bactericidal test. However, the true predictive value of B-1 cells and NAbs for the health status and immunocompetence of dairy cattle remains to be established. Proteomics turned out to be a useful approach for identifying potential new biomarkers for health and disease in milk of cows. Application and further development of their predictive capacity is dependent on the availability of robust, sensitive and quantitative assays. This project was part of the “Resilient Cattle” project providing biological samples and essential data on the health status during respective lactation periods of individual dairy cows. The impact of this research now requires translation into management tools and principles for the individual farmer impacting on the overall health status and economic performance of his herd of dairy cattle.

Supplementary data: Combined biotic and abiotic stress resistance in tomato
Kissoudis, C. ; Chowdhury, Rawnaq ; Heusden, A.W. van; Wiel, C.C.M. van de; Finkers, H.J. ; Visser, R.G.F. ; Bai, Y. ; Linden, C.G. van der - \ 2016
solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysisplant breeding - salt tolerance
Abiotic and biotic stress factors are the major constrains for the realization of crop yield potential. As climate change progresses, the spread and intensity of abiotic as well as biotic stressors is expected to increase, with increased probability of crops being exposed to both types of stress. Shielding crops from combinatorial stress requires a better understanding of the plant’s response and its genetic architecture. In this study, we evaluated resistance to salt stress, powdery mildew and to both stresses combined in tomato, using the S. habrochaites LYC4 introgression line (IL) population. The IL population segregated for both salt stress tolerance and powdery mildew resistance. Using SNP array marker data, QTLs were identified for salt tolerance as well as Na+ and Cl- accumulation. Salt stress increased the susceptibility of the population to powdery mildew in an additive manner. Phenotypic variation for disease resistance was reduced under combined stress as indicated by the coefficient of variation (CV). No correlation was found between disease resistance and Na+ and Cl- accumulation under combined stress Most genetic loci were specific for either salt stress tolerance or powdery mildew resistance. These findings increase our understanding of the genetic regulation of responses to abiotic and biotic stress combinations and can provide leads to more efficiently breeding for tomatoes and other crops with a high level of disease resistance while maintaining their performance in combination with abiotic stress.
Supplementary data: Responses to combined abiotic and biotic stress in tomato are governed by stress intensity and mechanism of resistance
Kissoudis, C. ; Sri Sunarti, Sri ; Wiel, C.C.M. van de; Visser, R.G.F. ; Linden, C.G. van der; Bai, Y. - \ 2016
solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysisplant breeding - salt tolerance
Stress conditions in agricultural ecosystems can occur in variable intensities. Different resistance mechanisms to 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 (PM) resistance and overall performance of tomato introgression lines with contrasting levels of partial resistance, as well as isogenic lines carrying the PM resistance genes Ol-1 (associated with slow Hypersensitivity Response; HR), ol-2 (a mlo mutant associated with papilla formation) and Ol-4 (a R gene associated with fast HR). PM 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 with accelerated cell death-like senescence. On the contrary, severe salt stress (150mM) reduced disease symptoms. Na+ and Cl- accumulation in the leaves was linearly related to the decreased pathogen growth under severe stress, suggesting a more direct role for the salt in suppressing PM growth. 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 of the variety Moneymaker (MM) and the NIL Ol-1 was associated with the induction of ethylene and jasmonic acid pathway genes as well as of the cell wall invertase gene LIN6. These results highlight the significance of stress severity and resistance type on the plant’s performance under abiotic and biotic stress combination.
Supplementary data: Hormone signalling regulation of tomato response to combined biotic and abiotic stress
Kissoudis, C. ; Sri Sunarti, Sri ; Wiel, C.C.M. van de; Visser, R.G.F. ; Linden, C.G. van der; Bai, Y. - \ 2016
solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysisplant breeding - salt tolerance
Plant hormones are paramount to plant adaptation to changing environmental conditions and interactions with microorganisms. There is currently limited knowledge on their significance in the response to stress combination. Using near isogenic lines (NILs) that carry the Ol-1, ol-2 and Ol-4 gene for resistance to tomato powdery mildew caused by Oidium neolycopersici, this study focused on the responses of these NILs to powdery mildew and salt stress combination. In these NILs, marker genes for monitoring hormonal pathways showed differential expression pattern upon powdery mildew infection. Further by crossing these NILs with tomato mutants notabilis (ABA-deficient), defenseless1 (JA-deficient) and epinastic (ET overproducer) the cross-talk among hormonal pathways was further investigated. Among the mutants, epinastic resulted in increased susceptibility of NIL-Ol-1 and breakdown of NIL-ol-2 resistance, accompanied by reduced callose deposition, effects that were more pronounced under combination with salt stress. On the other hand notabilis, resulting in H2O2 overproduction greatly reduced susceptibility of NIL-Ol-1 under combined stress accompanied however by heightened sensitivity to salt stress. Callose deposition reduction led to partial resistance breakdown in NIL-ol-2 which was reversed under combined stress. NIL-Ol-4 resistance remained robust across all mutant and treatment combinations. We discuss the critical role that hormone signalling appears to have for the outcome of combined stress and powdery mildew in terms of resistance and plant fitness integrating observations from physiological, histochemical and gene expression analyses. These significant insights obtained extend our understanding of hormonal regulation of combined stress responses and can aid in narrowing down targets for improving crop performance under stress combinations.
Supplementary data: Roles and contribution of tomato WRKY genes to salt stress and powdery mildew resistance
Kissoudis, C. ; Gao, D. ; Pramanik, Dewi ; Birhanu, Mengistu ; Wiel, C.C.M. van de; Visser, R.G.F. ; Bai, Y. ; Linden, C.G. van der - \ 2016
solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysisplant breeding - salt tolerance
WRKY is a transcription factor family unique to plants with diverse functions in defense pathways, abiotic stress tolerance and developmental programs. Family members are characterized by the conserved WRKY domain and significant sequence variation in the remainder of the protein, which is translated into distinct functions even for closely related genes. We utilized the extensive functional characterization of the Arabidopsis thaliana WRKY family to identify tomato homologues of Arabidopsis WRKY genes that are involved in defense responses (AtWRKY 11, 29, 48, 70 and 72). In total 13 tomato WRKY homologues were identified for these genes, of which 9 were successfully over-expressed, and 12 stably silenced via RNAi in transgenic tomato lines. The transgenic lines were evaluated for their response to salt stress, powdery mildew resistance and the combination of these stresses. Lines overexpressing SlWRKY11 and SlWRKY23, and RNAi lines of SlWRKY7 and SlWRKY9 showed both increased biomass and improved salt tolerance. For SlWRKY11 and SlWRKY23 overexpression (OE) lines, this was accompanied by a moderate increase in oxidative stress tolerance. The SlWRKY6-OE line showed strongly improved salt stress tolerance, but a growth penalty under control conditions. Exceptional phenotypes were observed for the SlWRKY10-OE line (stunted growth) and the RNAi line SlWRKY23-RNAi (necrotic symptoms), but these phenotypes were partly restored to normal under salt stress. Both these lines exhibited increased resistance to powdery mildew, but this was compromised when the plants were put under salt-stress as well. Important functions for tomato WRKY genes were revealed in both the abiotic and biotic stress response and several genes should be further explored to elucidate their downstream regulatory functions that lead to increased stress tolerance.
Ontwikkeling systeemaanpak meeldauw
Hofland-Zijlstra, J.D. ; Breeuwsma, S.J. ; Noordam, Marianne - \ 2016
- 1 p.
biologische bestrijding - tuinbouw - glastuinbouw - bestrijdingsmethoden - meeldauw - kasproeven - potplanten - tomaten - gerbera - rosaceae - ziekteresistentie - biological control - horticulture - greenhouse horticulture - control methods - mildews - greenhouse experiments - pot plants - tomatoes - disease resistance
Doel van dit onderzoek is het ontwikkelen van een systeemaanpak voor de beheersing van meeldauw door de inzet van groene producten (laag-risico profiel) die een bijdrage leveren aan het versterken van natuurlijke afweerreacties. Poster van het PlantgezondheidEvent 2016.
Resistentieveredeling - Resistentieveredeling in de praktijk : Kennisclip Bogo-project e-learning
Hop, M.E.C.M. - \ 2016
Groen Kennisnet
resistentieveredeling - overblijvende planten - boomkwekerijen - ziekteresistentie - selectiemethoden - vasteplantenkwekerijen - plantenveredeling - gewasbescherming - lesmaterialen - resistance breeding - perennials - forest nurseries - disease resistance - selection methods - perennial nurseries - plant breeding - plant protection - teaching materials
Deze kennisclip maakt onderdeel uit van de lesmodule Resistentie Veredeling van het CIV T&U.
Resistentieveredeling - Stressfactoren en verschillende typen belagers : Kennisclip Bogo-project e-learning.
Hop, M.E.C.M. - \ 2016
Groen Kennisnet
resistance breeding - stress factors - plant breeding - selection methods - disease resistance - plant protection - teaching materials - detection - pathogens - resistentieveredeling - stressfactoren - plantenveredeling - selectiemethoden - ziekteresistentie - gewasbescherming - lesmaterialen - detectie - pathogenen
Deze kennisclip maakt onderdeel uit van de lesmodule Resistentie Veredeling van het CIV T&U.
Resistentieveredeling - Overerving van resistentie : Kennisclip Bogo-project e-learning
Hop, M.E.C.M. - \ 2016
Groen Kennisnet
resistance breeding - disease resistance - genetic resistance - partial resistance - plant genetics - plant protection - teaching materials - resistentieveredeling - ziekteresistentie - genetisch bepaalde resistentie - partiële resistentie - plantengenetica - gewasbescherming - lesmaterialen
Deze kennisclip maakt onderdeel uit van de lesmodule Resistentie Veredeling van het CIV T&U.
Resistentieveredeling - Verdedigingsmechanisme : Kennisclip Bogo-project e-learning
Hop, M.E.C.M. - \ 2016
Groen Kennisnet
resistance breeding - susceptibility - resistance - tolerance - host pathogen interactions - plant protection - teaching materials - disease resistance - resistentieveredeling - vatbaarheid - weerstand - tolerantie - gastheer-pathogeen interacties - gewasbescherming - lesmaterialen - ziekteresistentie
Deze kennisclip maakt onderdeel uit van de lesmodule Resistentie Veredeling van het CIV T&U.
Breeding against infectious diseases in animals
Rashidi, H. - \ 2016
University. Promotor(en): Johan van Arendonk, co-promotor(en): Herman Mulder; P.K. Mathur. - Wageningen University - ISBN 9789462576452 - 179 p.
livestock - infectious diseases - animal breeding - selective breeding - disease resistance - tolerance - genetic variation - breeding value - genetic correlation - traits - genomics - animal genetics - vee - infectieziekten - dierveredeling - selectief fokken - ziekteresistentie - tolerantie - genetische variatie - fokwaarde - genetische correlatie - kenmerken - genomica - diergenetica

Infectious diseases in farm animals are of major concern because of animal welfare, production costs, and public health. Farms undergo huge economic losses due to infectious disease. The costs of infections in farm animals are mainly due to production losses, treatment of infected animals, and disease control strategies. Control strategies, however, are not always successful. Selective breeding for the animals that can mount a defence against infection could therefore be a promising approach. Defensive ability of an animal has two main mechanisms: resistance (ability to control the pathogen burden) and tolerance (ability to maintain performance when pathogen burden increases). When it is difficult to distinguish between resistance and tolerance, defensive ability is measured as resilience that is the ability to maintain performance during a disease outbreak regardless of pathogen burden. Studies have focused on the genetics of resistance and resilience with little known about the genetics of tolerance and its relationship with resistance and resilience. The objectives of this thesis were to: 1) estimate the genetic variation in resistance, tolerance, and resilience to infection in order to assess the amenability of these traits for selective breeding in farm animals, 2) estimate the genetic correlation between resistance, tolerance and resilience and 3) detect genomic regions associated with resistance, tolerance, and resilience.

In chapter 2, we studied the variation among sows in response to porcine reproductive and respiratory syndrome (PRRS). First a statistical method was developed to detect PRRS outbreaks based on reproduction records of sows. The method showed a high sensitivity (78%) for disease phases. Then the variation of sows in response to PRRS was quantified using 2 models on the traits number of piglets born alive (NBA) and number of piglets born dead (LOSS): 1) bivariate model considering the trait in healthy and disease phases as different traits, and 2) reaction norm model modelling the response of sows as a linear regression of the trait on herd-year-week estimates of NBA. Trait correlations between healthy and disease phases deviated from unity (0.57±0.13 – 0.87±0.18). The repeatabilities ranged from 0.07±0.027 to 0.16±0.005. The reaction norm model had higher predictive ability in disease phase compared to the bivariate model.

In chapter 3 we studied 1) the genetic variation in resistance and tolerance of sheep to gastrointestinal nematode infection and 2) the genetic correlation between resistance and tolerance. Sire models on faecal nematode egg count (FEC), IgA, and pepsinogen were used to study the genetic variation in resistance. Heritability for resistance traits ranged from 0.19±0.10 to 0.59±0.20. A random regression model was used to study the reaction norm of sheep body weight on FEC as an estimate of tolerance to nematode infection. We observed a significant genetic variance in tolerance (P<0.05). Finally a bivariate model was used to study the genetic correlation between resistance and tolerance. We observed a negative genetic correlation (-0.63±0.25) between resistance and tolerance.

In chapter 4, we studied the response to selection in resistance and tolerance when using estimated breeding values for resilience. We used Monte Carlo simulation to generate 100 half-sib families with known breeding values for resistance (pathogen burden) and tolerance. We used selection index theory to predict response to selection for resistance and tolerance: 1) when pathogen burden is known and selection is based on true breeding values for resistance and tolerance and 2) when pathogen burden is unknown and selection is based on estimated breeding values for resilience. Using EBV for resilience in absence of records for pathogen burden resulted in favourable responses in resistance and tolerance to infections, with more emphasis on tolerance than on resistance. However, more genetic gain in resistance and tolerance could be achieved when pathogen burden was known.

In chapter 5 we studied genomics regions associated with resistance, resilience, and tolerance to PRRS. Resistance was modelled as sire effect on area under the PRRS viremia curve up to 14 days post infection (AUC14). Resilience was modelled as sire effects on daily growth of pigs up to 28 days post infection (ADG28). Tolerance was modelled as the sire effect on the regression of ADG28 on AUC14. We identified a major genomics region on chromosome 4 associated with resistance and resilience to PRRS. We also identified genomics regions on chromosome 1 associated with tolerance to PRRS.

In the general discussion (chapter 6) I discussed: 1) response to infection as a special case of genotype by environment interaction, 2) random regression model as a statistical tool for studying response to disease, 3) advantages and requirements of random regression models, and 4) selective breeding of farm animals for resistance, tolerance, and resilience to infections. I concluded that random regression is a powerful approach to estimate response to infection in animals. If the adequate amount of data is available random regression model could estimate breeding values of animals more accurately compared to other models. I also concluded that before including resistance and tolerance into breeding programs, breeders should make sure about the added values of including these traits on genetic progress. Selective breeding for resilience could be a pragmatic approach to simultaneously improve resistance and tolerance.

Role of MLO genes in susceptibility to powdery mildew in apple and grapevine
Pessina, Stefano - \ 2016
University. Promotor(en): Richard Visser, co-promotor(en): Henk Schouten; M. Malnoy; Yuling Bai. - Wageningen : Wageningen University - ISBN 9789462576209 - 222 p.
malus domestica - apples - vitis vinifera - grapes - plant pathogenic fungi - podosphaera leucotricha - erysiphe necator - disease resistance - susceptibility - genes - gene expression - gene knock-out - resistance breeding - appels - druiven - plantenziekteverwekkende schimmels - ziekteresistentie - vatbaarheid - genen - genexpressie - inactivering van genen - resistentieveredeling

Powdery mildew (PM) is a major fungal disease that threatens thousands of plant species. PM is caused by Podosphaera leucotricha in apple and Erysiphe necator in grapevine. Powdery mildew is controlled by frequent applications of fungicides, having negative effects on the environment, and leading to additional costs for growers. To reduce the amount of chemicals required to control this pathogen, the development of resistant apple and grapevine varieties should become a priority.

PM pathogenesis is associated with up-regulation of specific MLO genes during early stages of infection, causing down-regulation of plant defense pathways. These up-regulated genes are responsible for PM susceptibility (S-genes) and their knock-out causes durable and broad-spectrum resistance. All MLO S-genes of dicots belong to the phylogenetic clade V. In grapevine, four genes belong to clade V. VvMLO7, 11 and 13 are up-regulated during PM infection, while VvMLO6 is not.

Chapter 2 reports the genome-wide characterization and sequence analysis of the MLO gene family in apple, peach and woodland strawberry, and the isolation of apricot MLO homologs. Twenty-one homologues were found in apple, 19 in peach and 17 in woodland strawberry. Evolutionary relationships between MLO homologs were studied and syntenic blocks constructed. Candidate genes for causing PM susceptibility were inferred by phylogenetic relationships with functionally characterized MLO genes and, in apple, by monitoring their expression following inoculation with the PM causal pathogen P. leucotricha. In apple, clade V genes MdMLO11 and 19 were up-regulated, whereas the two other members of clade V, MdMLO5 and 7, were not up-regulated. The clade VII gene MdMLO18 was also up-regulated upon P. leucotricha infection.

Chapter 3 reports the knock-down, through RNA interference, of MdMLO11 and 19, as well as complementation of the mutant phenotype by expression of the MdMLO18 gene in the Arabidopsis thaliana triple mlo mutant Atmlo2/6/12. The knock-down of MdMLO19 resulted in a reduction of PM disease severity up to 75%, whereas the knock-down of MdMLO11, alone or combined with MdMLO19, did not cause any reduction or additional reduction of susceptibility compared to MdMLO19 alone. Complementation by MdMLO18 did not restore susceptibility. Cell wall appositions (papillae), a response to PM infection, were found in both susceptible plants and PM resistant plants where MdMLO19 was knocked-down, but were larger in resistant lines. The expression analysis of 17 genes related to plant defense, and quantification of phenolic metabolites in resistant lines revealed line-specific changes compared to the control.

Chapter 4 evaluates the presence of non-functional alleles of the MdMLO19 S-gene in apple germplasm. The screening of the re-sequencing data of 63 apple genotypes led to the identification of 627 SNP in five MLO genes (MdMLO5, MdMLO7, MdMLO11, MdMLO18 and MdMLO19). Insertion T-1201 in MdMLO19 caused the formation of an early stop codon, resulting in a truncated protein lacking 185 amino-acids and the calmodulin-binding domain. The presence of the insertion was evaluated in a collection of 159 apple genotypes: it was homozygous in 53 genotypes, 45 of which were resistant or very resistant to PM, four partially susceptible and four not assessed. These results strongly suggest that this insertion is causative for the observed PM resistance. The absence of a clear fitness cost associated to the loss-of-function of MdMLO19, might have contributed to the high frequency of the mutation in breeding germplasm and cultivars. Among the genotypes containing the homozygous insertion, ‘McIntosh’ and ‘Fuji’ are commonly used in apple breeding. After barley and tomato, apple is the third species with a reported natural non-functional mlo allele in its germplasm, with the important difference that the allele is present in a relatively large number of apple genotypes, most of which not related to each other.

Chapter 5 reports the knock-down through RNA interference of four grapevine MLO genes, all members of clade V. VvMLO7, 11 and 13 are up-regulated in early stages of infection, whereas VvMLO6 is not responsive to the pathogen. Knock-down of VvMLO6, 11 and 13, alone or combined, did not decrease PM severity, whereas the knock-down of VvMLO7, alone or in combination with VvMLO6 and VvMLO11, caused a reduction of severity of 77%. Cell wall appositions (papillae), a response to PM attack, were present in both resistant and susceptible lines, but were larger in resistant lines. Thirteen genes involved in defense were less up-regulated in resistant plants, highlighting the reduction of PM disease severity.

In Chapter 6 we discuss the results presented in this thesis. The pivotal role of MLO genes in the interaction of PM pathogens with apple and grapevine is described and further experiments aimed at addressing open questions are proposed. The results described in this thesis open interesting avenues in MLO genes research, particularly the finding that a natural mlo mutation in apple appeared to be more common than expected. This mutation is directly applicable in marker assisted breeding for durable PM resistance in apple.

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