Annelies E. H. M. Loonen

Reduced Position Effect in Mature Transgenic Plants Conferred by the Chicken Lysozyme Matrix-Associated Region

Matrix-associated regions may be useful for studying the role of chromatin architecture in transgene activity of transformed plants. The chicken lysozyme A element was shown to have specific affinity for tobacco nuclear matrices, and its influence on the variability of transgene expression in tobacco plants was studied. T-DNA constructs in which this element flanked either the [beta]-glucuronidase (GUS) reporter gene or both reporter and selection gene were introduced in tobacco. The variation in GUS gene activity was reduced significantly among mature first-generation transgenic plants carrying the A element. Average GUS activity became somewhat higher, but the maximum attainable level of gene expression was similar for all three constructs. Transient gene expression assays showed that the A element did not contain general enhancer functions; therefore, its presence seemed to prevent the lower levels of transgene expression. The strongest reduction in variability was found in plants transformed with the construct carrying the A elements at the borders of the T-DNA. In this population, expression levels became copy number dependent. The presence of two A elements in the T-DNA did not interfere with meiosis.

The Presence of a Chromatin Boundary Appears to Shield a Transgene in Tobacco from RNA Silencing

We present isogenic transgenic tobacco lines that carry at a given chromosomal position a β-glucuronidase (GUS) reporter gene either with or without the presence of the matrix-associated region known as the chicken lysozyme A element. Plants were generated with the Cre-lox site–specific recombination system using heterospecific lox sites. Analysis of GUS gene expression in plant populations demonstrates that the presence of the A element can shield against RNA silencing of the GUS gene. Protection was observed in two of three independent tobacco transformants. Plants carrying an A element 5′ of the GUS gene always had stable GUS activity, but upon removal of this A element, the GUS gene became silenced over time in two lines, notably when homozygous.

Powdery mildew resistance in tomato by impairment of SlPMR4 and SlDMR1.

Genetic dissection of disease susceptibility in Arabidopsis to powdery and downy mildew has identified multiple susceptibility (S) genes whose impairment results in disease resistance. Although several of these S-genes have been cloned and characterized in more detail it is unknown to which degree their function in disease susceptibility is conserved among different plant species. Moreover, it is unclear whether impairment of such genes has potential in disease resistance breeding due to possible fitness costs associated with impaired alleles. Here we show that the Arabidopsis PMR4 and DMR1, genes encoding a callose synthase and homoserine kinase respectively, have functional orthologs in tomato with respect to their S-gene function. Silencing of both genes using RNAi resulted in resistance to the tomato powdery mildew fungus Oidium neolycopersici. Resistance to O. neolycopersici by SlDMR1 silencing was associated with severely reduced plant growth whereas SlPMR4 silencing was not. SlPMR4 is therefore a suitable candidate gene as target for mutagenesis to obtain alleles that can be deployed in disease resistance breeding of tomato.

Frequent spontaneous deletions of Ri T-DNA in Agrobacterium rhizogenes transformed potato roots and regenerated plants.

The presence of T-DNA was examined by Southern blot analysis in 16 regenerated shoot lines derived from 6 Agrobacterium rhizogenes-transformed root clones of Solanum tuberosum L. cv. Bintje.TR-DNA, present in regenerated shoot lines from 3 out of 6 root clones was correlated with the presence of opines. One root clone produced opines up to 2.5 years of subculture. However, plant regeneration from and prolonged subculturing of this root clone resulted in loss of opine synthesis, caused by deletion of TR-DNA.TL-DNA inserted at 1 to 5 independent loci was found in 14 of the 16 shoot lines. Surprisingly, 1 to 2 additional insertions next to similar insertions of TL-DNA were found in shoot lines from the same root clone (named ‘sister’ shoot lines) in 2 out of 4 root clones. Nevertheless, this did not result in gross phenotypic variation between sister shoot lines. Another root clone regenerated 1 shoot line with an Ri phenotype, containing 1 insertion of TL-DNA, and 2 shoot lines with a normal Bintje phenotype without TL-DNA. The 5th root clone showed no difference between sister shoot lines and the 6th root clone produced only 1 shoot line.We conclude that during prolonged root culture and during shoot regeneration from root clones deletion of TL- and TR-DNA insertions can occur. The significance of the frequency of deletion of T-DNA of the Ri plasmid is discussed.

Activity of the promoter of the Lhca3.St.1 gene, encoding the potato apoprotein 2 of the light-harvesting complex of photosystem I, in transgenic potato and tobacco plants

We have isolated cDNA and genomic clones for the potato (Solanum tuberosum) apoprotein 2 of the light harvesting complex of Photosystem I, designated Lhca3.St.1. The protein shows all characteristics of the family of chlorophyll a/b-binding proteins. Potato Lhca3.1 gene expression occurs predominantly in leaves, and is transcriptionally regulated by light. One gene copy is present per haploid genome. The sequence of the 5′ upstream region was determined. Most boxes identified in the promoter sequences of genes whose expression is light-regulated recur in the Lhca3.St.1 sequence. Functional analyses of the Lhca3.St.1 promoter and two deletion derivatives in transgenic potato transformed with a promoter-GUS fusion show high promoter activity in leaves and other green parts of the plant, which depends on light. Activity is absent in roots and potato tubers. The 500 bp promoter fragment is as active as the full 2.0 kb sequence, showing that all regulatory elements are present on the smallest deletion derivative. In transgenic tobacco (Nicotiana tabacum) plants carrying the largest promoter derivative a similar distribution of activity is found. Promoter activity is not restricted to the phloem, but also prominent in the xylem of the young stem, which contrasts with promoters of other photosynthesis-associated genes.

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

Multiple susceptibility genes (S), identified in Arabidopsis, have been shown to be functionally conserved in crop plants. Mutations in these S genes result in resistance to different pathogens, opening a new way to achieve plant disease resistance. The aim of this study was to investigate the role of Defense No Death1 (DND1) in susceptibility of tomato and potato to late blight (Phytophthora infestans). In Arabidopsis, the dnd1 mutant has broad-spectrum resistance against several fungal, bacterial, and viral pathogens. However this mutation is also associated with a dwarfed phenotype. Using an RNAi approach, we silenced AtDND1 orthologs in potato and tomato. Our results showed that silencing of the DND1 ortholog in both crops resulted in resistance to the pathogenic oomycete P. infestans and to two powdery mildew species, Oidium neolycopersici and Golovinomyces orontii. The resistance to P. infestans in potato was effective to four different isolates although the level of resistance (complete or partial) was dependent on the aggressiveness of the isolate. In tomato, DND1-silenced plants showed a severe dwarf phenotype and autonecrosis, whereas DND1-silenced potato plants were not dwarfed and showed a less pronounced autonecrosis. Our results indicate that S gene function of DND1 is conserved in tomato and potato. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of DND1 orthologs, as well as additional S gene orthologs from Arabidopsis, to breed for resistance to pathogens in crop plants.

Analysis of the region in between two closely linked patatin genes: class II promoter activity in tuber, root and leaf

From a potato genomic library a phage lambda clone was isolated that carried nucleotide sequences of two patatin genes, thus demonstrating a close physical linkage between these two members of the patatin gene family. Sequence and restriction analysis showed the genes to be oriented in tandem. The more upstream gene was a pseudogene truncated at the 3′ end, whereas the downstream gene was a class II patatin gene. In addition to a 208 bp fragment also present in patatin class I promoters, the region in between both genes contained various direct repeats also found in other patatin genes. To study the promoter activity of this intergenic region, a 2.78 kb fragment was transcriptionally fused to the β-glucuronidase gene and reintroduced into potato cultivar Bintje. Histochemical analysis revealed expression in the outermost layer of cells of the cortex, in the tuber phellogen, in or around the root vascular system, and also in the abaxial phloem layer of the vascular bundle in leaves.

Activation tagging of ATHB13 in Arabidopsis thaliana confers broad-spectrum disease resistance

Powdery mildew species Oidium neolycopersici (On) can cause serious yield losses in tomato production worldwide. Besides on tomato, On is able to grow and reproduce on Arabidopsis. In this study we screened a collection of activation-tagged Arabidopsis mutants and identified one mutant, 3221, which displayed resistance to On, and in addition showed a reduced stature and serrated leaves. Additional disease tests demonstrated that the 3221 mutant exhibited resistance to downy mildew (Hyaloperonospora arabidopsidis) and green peach aphid (Myzus persicae), but retained susceptibility to bacterial pathogen Pseudomonas syringae pv tomato DC3000. The resistance trait and morphological alteration were mutually linked in 3221. Identification of the activation tag insertion site and microarray analysis revealed that ATHB13, a homeodomain-leucine zipper (HD-Zip) transcription factor, was constitutively overexpressed in 3221. Silencing of ATHB13 in 3221 resulted in the loss of both the morphological alteration and resistance, whereas overexpression of the cloned ATHB13 in Col-0 and Col-eds1-2 backgrounds resulted in morphological alteration and resistance. Microarray analysis further revealed that overexpression of ATHB13 influenced the expression of a large number of genes. Previously, it was reported that ATHB13-overexpressing lines conferred tolerance to abiotic stress. Together with our results, it appears that ATHB13 is involved in the crosstalk between abiotic and biotic stress resistance pathways.

Silencing of six susceptibility genes results in potato late blight resistance

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

Down-regulation of acetolactate synthase compromises Ol-1- mediated resistance to powdery mildew in tomato

BackgroundIn a cDNA-AFLP analysis comparing transcript levels between powdery mildew (Oidium neolycopersici)-susceptible tomato cultivar Moneymaker (MM) and near isogenic lines (NILs) carrying resistance gene Ol-1 or Ol-4, a transcript-derived fragment (TDF) M11E69-195 was found to be present in NIL-Ol-1 but absent in MM and NIL-Ol-4. This TDF shows homology to acetolactate synthase (ALS). ALS is a key enzyme in the biosynthesis of branched-chain amino acids valine, leucine and isoleucine, and it is also a target of commercial herbicides.ResultsThree ALS homologs ALS1, ALS2, ALS3 were identified in the tomato genome sequence. ALS1 and ALS2 show high similarity, whereas ALS3 is more divergent. Transient silencing of both ALS1 and ALS2 in NIL-Ol-1 by virus-induced gene silencing (VIGS) resulted in chlorotic leaf areas that showed increased susceptibility to O. neolycopersici (On). VIGS results were confirmed by stable transformation of NIL-Ol-1 using an RNAi construct targeting both ALS1 and ALS2. In contrast, silencing of the three ALS genes individually by RNAi constructs did not compromise the resistance of NIL-Ol-1. Application of the herbicide chlorsulfuron to NIL-Ol-1 mimicked the VIGS phenotype and caused loss of its resistance to On. Susceptible MM and On-resistant line NIL-Ol-4 carrying a nucleotide binding site and leucine rich repeat (NB-LRR) resistance gene were also treated with chlorsulfuron. Neither the susceptibility of MM nor the resistance of NIL-Ol-4 was affected.ConclusionsALS is neither involved in basal defense, nor in resistance conferred by NB-LRR type resistance genes. Instead, it is specifically involved in Ol-1-mediated resistance to tomato powdery mildew, suggesting that ALS-induced change in amino acid homeostasis is important for resistance conferred by Ol-1.