The role of RNA silencing in the Verticillium wilt disease

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Marieke Leijten

Role of RNA silencing in Verticillium

wilt disease

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Role of RNA silencing in Verticillium

wilt disease

Version: 1.0

Date: 05.06.2013

School data Avans Hogescholen

Address: Lovensdijkstraat 61-63

4818 AJ Breda

Department: Academy of the Technology of Health and Environment

Student: Marieke Leijten mjqmleij@avans.nl

Supervisor: Nicole van den Braak npwcj.vandenbraak@avans.nl

Internship data

Department: Phytopathology

Address: Droevendaalsesteeg 1

Building 107

6708 PB Wageningen

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5 J un e 2 0 1 3 3 The role of RNA silencing in the Verticillium wilt diseae

Abstract

Plant pathogens play an important role in the food and feed production, among these vascular wilts are the most devastating diseases worldwide. Verticillium dahliae is one of these plant pathogens, which can infect 200-300 plant species and is known to cause a lot of crop losses every year. Despite the economic importance of Verticillium, the molecular basis of Verticillium wilt disease is relatively unknown.

RNA silencing plays a crucial role in the interaction between Arabidopsis thaliana and Verticillium dahliae. RNA silencing is a mechanism that makes use of small non-coding RNAs (small-RNAs) to regulate gene

expression in a sequence specific manner. In this project gene targets of small-RNAs were identified and the biological relevance during Verticillium wilt disease was confirmed.

First we verified the putative role of 47 gene targets during Verticillium wilt disease. We analysed Arabidopsis T-DNA insertion mutants that were mutated for the specific target gene, 53 different mutant lines were tested in total. We also determined by PCR if these mutants were mutated and carried the T-DNA insertion on both chromosomes in the specified gene targets. Only for 26 of the 47 predicted homozygous lines we could confirm the correct presence of the T-DNA on both chromosomes. From this we can conclude that the putative homozygous lines are often heterozygous, because only one of the both chromosomes contained the T-DNA, or incorrect, the T-DNA insertion is located elsewhere on the chromosome or not present at all (a wild-type, Col-0, instead of a mutant plant). Still a fair amount of 25 of the tested mutant lines showed an increase in Verticillium resistance, when compared to the wild-type Col-0, providing the evidence that the selected genes are involved in Verticillium colonization.

The identified target genes were originally selected based on their transcript accumulation in sgs2-1 mutant when compared to Col-0 after Verticillium infection. We could confirm the regulation of 25 transcripts of the target genes in sgs2-1 mutant and Col-0 at 3, 6 and 9 days post Verticillium infection. The trend of the transcript regulation in this biological control experiment was comparable with the original RNA sequencing data. Accumulation of a specific transcript in the RNA sequencing analysis showed an increase in expression detected by qPCR in the sgs2-1 mutant when compared to the Col-0, both after the infection with Verticillium. Finally, for a few candidate genes we have also confirmed the presence of the specific small-RNAs that target them.

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Theoretical background

Plant-pathogen model system used for our studies

The plant fungus Verticillium dahliae is able of infecting 200-300 plant species, including fruit and nut trees, vegetables, forest trees and herbaceous ornamentals. The fungus is causing Verticillium wilt disease and this disease leads to billions of dollars in annual crop losses worldwide. The fungus is infecting the host plants through their vascular system, so it cannot be reached by many fungicides, only growing Verticillium dahliae resistant crops are capable of preventing the disease (López-Escudero and Blanco-López 2005; Fradin, Zhang et al. 2009; Klosterman, Atallah et al. 2009).

Verticillium dahliae has a life cycle, this cycle is shown in Figure 1. The cycle starts with inactive microsclerotia which can remain in the soil for up to 14 years (Klosterman, Atallah et al. 2009). The

microsclerotia are activated by plant root exudates, these will produce hyphae which are growing to extent towards the host roots. The fungus will reach the conducting xylem vessels and will produce conidiospores, these are carried upwards through the plant with the water flow. This results in a vascular colonization by the fungus and of the host defence response to close infected xylem vessels, the plant starts to wilt (Agrios 2005; López-Escudero and Blanco-López 2005; Fradin and Thomma 2006; Klosterman, Atallah et al. 2009).

During this research the model plant Arabidopsis thaliana (from here termed as Arabidopsis) or mouse-ear cress is used, this plant is a member of the mustard family (Cruciferae or Brassicaceae) and is native to Europe, Asia, North western Africa and North America. In this research the accepted standard Columbia is used (Meinke, Cherry et al. 1998). Arabidopsis is a small popular plant with scientist and the sequence is known since 2000 (Arabidopsis Genome 2000; Glick, Pasternak et al. 2010). The plant is a diploid plant which means it has two chromosomes, when tested for homozygosis they both should have a mutation. The plant has one of the smallest genomes of any flowering plant (7x107 bp), the size is similar to the yeast genome (1.5x107 bp). The plant can grow from seed to mature seed within 6 weeks and can grow on petri plates, pots located in a greenhouse or under fluorescent lights in the laboratory (Meinke, Cherry et al. 1998; Arabidopsis Genome 2000). Figure 2 shows the different growth stages of Arabidopsis (Meinke, Cherry et al. 1998; Johansson, Olsson et al. 2004).

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Figure 2: Arabidopsis thaliana from early growth to fully grown plants, including the flowers, which will be formed in a later stage of growth

Figure 1: Verticillium wilt disease cycle (Berlanger and Powelson 2000)

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RNA silencing

RNA silencing was discovered in plants 15-20 years ago. The discovery was done during the course of transgenic experiments that led to silencing of the introduced transgene. RNA silencing refers collectively to diverse RNA-based processes, which eventually inhibit all results in sequence-specific manner of gene expression. This can either be at the transcription mRNA-stability or at translational levels. RNase- III-ribonuclease Dicer-like (DCL) proteins generate small-RNAs. DCLs function with Argonaute (AGO) proteins in RNA-induced silencing complex. During RNA silencing small-RNAs are produced out of ds-RNA and are 20-26 bp in length (Brodersen and Voinnet 2006; Vaucheret 2006; Kuang, Padmanabhan et al. 2009).

For RNA silencing in Arabidopsis there are currently four different pathways known, based on four different DCL proteins. Each pathway produces different types of small-RNAs, the most studied are the mi-RNAs (Figure 3, pathway on the left side). During the research we will focus on the trans acting small-RNA (tasi-RNA) producing-pathway (Figure 3, pathway on the right side). Figure 3 shows that both pathways, the mi-RNA pathway and the tasi-RNA pathway are linked (Vaucheret 2006; Eamens, Wang et al. 2008). mi-RNAs are produced by the DCL complex from imperfect RNA hairpins. Tasi-RNAs are generated from non-coding transcripts, named TAS transcripts (Hamilton, Voinnet et al. 2002; Matzke and Birchler 2005; Garcia 2008).

Besides the DCL complex and TAS transcript the production of tasi-RNAs also depends on a functional RNA-dependent RNA polymerase (RdRP), which is a polymerase that can synthesize ds-RNA from ss-RNA templates to initiate or amplify the RNA silencing reaction (Hamilton, Voinnet et al. 2002; Matzke and Birchler 2005; Garcia 2008).

The Arabidopsis genome contains four families of tasi-RNA-generating TAS genes. TAS1A/B/C and TAS2 are cleaved by miR173,-TAS3A/B/C are cleaved by miR390 and TAS4 is cleaved by miR828 (Yoshikawa, Peragine et al. 2005; Rajagopalan, Vaucheret et al. 2006).

Figure 3: mi-RNA and tasi-RNA pathways, adapted from (Eamens, Wang et al. 2008; Garcia 2008). The left panel shows the formation of mi-RNA hairpins resulting in mi-RNAs and the right panel the formation of tas-RNAs from the TAS3 precursor.

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Role of RNA silencing in Verticillium wilt disease

RNA silencing is involved in many and diverse processes, ranging from development to pathogen defence and a number of mi-RNAs have been linked to biotic stress. Plant RNA silencing is known to play a role during initiate immunity against viruses and bacteria (Lu, Kulkarni et al. 2006; Eamens, Wang et al. 2008; Chuck, Candela et al. 2009). In the host lab, a role for RNA silencing in defence against Verticillium was suggested,

because various Arabidopsis RNA silencing mutants display increased or reduced Verticillium resistance (Ellendorff, Fradin et al. 2009). Figure 4 shows the thirteen different Arabidopsis RNA silencing mutants that were tested for Verticillium colonization (Figure 4 is adapted from data that was published by: Ellendorf, Fradin et al. 2009). The Verticillium biomass was quantified 21 days post infection (dpi) and this level is indicative for an increased or reduced Verticillium resistance. The identified Verticillium biomass in the Col-0 plants was set to 100% and the bars in green indicated an increased susceptibility, while the red bars indicate an increased resistance against Verticillium. For most RNA silencing mutants the gene that is mutated and as such not expressed in these plants is known. For most except the suppressors of gene silencing (sgs) mutants the absence of the RNA silencing genes causes pleiotropic effects on general plant development. To exclude that the altered Verticillium growth is caused by pleiotropic, or developmental artefacts of the plant, the further focus was on the sgs-mutants. The sgs-mutants, do not show any additional phenotype besides increased Verticillium susceptibility.

The sgs-Arabidopsis mutants were identified in a forward screen by Mourrain et al (2000). The sgs1-1, sgs2-1 and sgs3-1 mutants are more susceptible to Verticillium (Figure 4). The corresponding gene sgs 1-1 is not identified yet (Mourrain, Beclin et al. 2000; Ellendorff, Fradin et al. 2009). SGS2 encodes for

RNA-dependent RNA polymerase 6 (RDR6) which is required for the biogenesis of tasi-RNAs (Mourrain, Beclin et al. 2000).

The trans-acting small-RNA (tasi-RNA) producing-pathway is introduced in the previous section. The production of tasi-RNAs depends besides on RDR6 also on SGS3 and DICER-LIKE 4 (DCL4) (Felippes and Weigel 2009).

We postulate that the lack of a specific group of tasi-RNAs and their up-regulated target genes causes the increased susceptibility to Verticillium within the sgs2-1 mutant. In this research we want to identify the tasi-RNAs and their cognate accumulated transcript targets that are absent during Verticillium colonization in the sgs2-1 mutant, because we speculate that they are the key for the basal defence against Verticillium in Col-0.

Figure 4: Verticillium biomass quantification of thirteen Arabidopsis RNA silencing mutants (Ellendorff, Fradin et al. 2009)

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Results

Transcript and small RNA isolation of Col-0 and sgs2-1 Arabidopsis plants after Verticillium

infection

To identify Arabidopsis transcripts (mRNA) and small-RNAs that are regulated during Verticillium

infection, deep parallel sequencing was performed. Two time points were selected, 3 and 6 days post infection (dpi) and mock treatment of the wild-type (Col-0) and sgs2-1 mutant. The transcriptomes were analysed by RNA sequencing at the Beijing Genomics Institute (BGI), while the small-RNAs library preparations and sequencing was outsourced to VERTIS Biotechnology AG in Germany.

Identification of the target genes

Figure 5 shows the selection scheme that was used to analyse and validate the set of identified small-RNAs and their cognate target genes for the involvement in basal defence against Verticillium. The small-small-RNAs that were identified by RNA sequencing from Col-0 and sgs2-1 plants 6 days post Verticillium infection were compared. Out of all the detectable small-RNAs, 344 were differential regulated between Col-0 versus sgs2-1 mutant 6 days after Verticillium infection. The plant version of UEA sRNA toolkit was used to predict the target genes of these 344 small RNA sequences (Moxon, Jing et al. 2008). 585 putative target genes were predicted, but only the transcripts of the target genes which had a negative correlation with the regulated small-RNAs suggest to be valid si-RNA target genes. Analysis of the expression patterns of the transcripts based on RNA sequencing data of the 585 putative target genes resulted in 32 genes that showed a negative expression correlation with the 35 regulated small-RNA. Attachment 4 shows the full list of small RNAs and negatively correlated tested target genes.

Three steps were performed after this selection to validate the relevance of the small-RNAs and their target genes during the Verticillium interaction. First the importance of the identified genes were studied during Verticillium colonization. Homozygous Arabiddopsis T-DNA insertion lines were ordered, these mutant lines carry a T-DNA insertion in the specified gene on both chromosomes, resulting in the absence of a functional transcript. If the homozygous lines were not available yet, they were generated. Step two was the confirmation of the regulation of the gene transcripts in a biological independent sample of Col-0 and sgs2-1 infection Verticillium plants. The third step was to confirm the presence and specific regulation of the small- RNAs by a PCR based method.

Figure 5: A schematic picture of the small-RNA and target gene selection

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Figure 7: Two examples of tested lines

N661036: shows a wild-type figure, because PCR products are formed with the gene primers. But it is also possible that the T-DNA primers would also work, so this mutant is termed as unknown. N652983: shows a heterozygous pattern, because PCR products are shown for both the T-DNA insertion combination and the primers to detect the intact gene.

Figure 6: Examples of two homozygous lines

The mutants display a T-DNA insertion within the same gene but on different locations. Both mutants are homozygous gene knock out mutants. This is shown by the PCR product which indicates the presence of the T-DNA. In the Col-0 plant the intact gene is present shown by the PCR product generated with the gene specific primers.

Generation of homozygous T-DNA lines

To confirm for all the putative homozygous knock out lines (as is indicated by the SALK institute) that the T-DNA insertion was present in both chromosomes and within the specific genes, a detection PCR was performed on the genomic DNA that was isolated from leaf material. This was done by detecting the T-DNA insertion with PCR and gel electrophoresis. Specific primers within the gene sequence that surrounded the T-DNA insertion site were generated. For the T-T-DNA insert a primer located at the left border was used. As a positive control Col-0 was used, the wild-type would only show a PCR product with the specific gene primer combination indicative for the intact none disrupted gene. The mutated genes will show a band for the left border primer combined with the right primer of the gene (from here on termed as T-DNA insertion combination). If the T-DNA was inserted within the gene on both chromosomes no PCR product should be generated with gene specific primers.

Some ordered lines were still segregating, for these lines first a pre-selection on plate with specific antibiotic was made. Plants that were resistant carried at least one T-DNA insertion, because the T-DNA contains the antibiotic resistant gene. After two weeks of growing in the climate chamber they were

transplanted in the soil and grown for another week. Leaf samples were taken and genomic DNA was isolated from these mutants and they were also tested for their putative homozygous T-DNA insertion.

Multiple collection of Arabidopsis T-DNA insertion lines are available. The T-DNA insertion lines were assayed for homozygosis from three different collections. In total 35 SALK lines were tested of which 15 were homozygous, 11 SAIL lines were tested of which 4 were potential homozygous and 6 GABI-KAT lines were tested of which 3 were homozygous.

In Figure 7 are two examples shown of two different mutants. One shows a wild-type pattern on the gel, the second mutant shows a heterozygous pattern. When bands were formed with the gene primers, the mutant was termed as wild-type. If the gel shows bands with the gene and the T-DNA insertion combination, this mutant was termed as heterozygous.

In Figure 6 are two mutants shown which are both homozygous. This is clear because there is only a band for the gene in the Col-0 background and only in the mutant sample we can detect the PCR fragment for the T-DNA insertion combination. If the mutated plant was heterozygous the plants were kept for seed production (Arabidopsis is a self-pollinator) and the seeds of the heterozygous mutant plants were screened again. One fourth of the next generation of the heterozygous plant is expected to be homozygous for the T-DNA insertion. Five to ten plants were tested for the T-DNA insertion on both chromosomes.

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Figure 9: Results of Verticillium infection 21 dpi Results of 3 tested mutants and Col-0. Plants are harvested 21 days post infection.

Figure 8: The biomass data after Verticillium infection harvested at 21 dpi

The baseline set at 1 indicate the biomass level of Col-0. Was the amount of the biomass lower than this line, the mutants were resistance. Was the amount of biomass higher than this line, the mutants were more susceptible.

Verticillium colonisation of mutated Arabidopsis lines

The Verticillium infection was performed on mutant plants of all selected genes to detect if a functional gene was required for the full Verticillium colonization. The mutants were photographed at 21 dpi and the genomic DNA was isolated to quantify the Verticillium biomass by quantitative PCR analysis. The Verticillium biomass is an indication for increase in susceptibility or resistance. The Verticillium biomass levels within the mutants were compared to the Col-0 infected plants. For the biomass quantification we used the detection of the Ave1 gene from Verticillium and the Rubisco Arabidopsis gene was used as an internal standard.

Figure 8 shows the graph of the mutant, some of the genes have two independent knockout mutants, meaning two independent T-DNA insertion lines in the same gene. The graph and table with all tested genes are shown in Attachment 3 and Attachment 5.

The mutants N681837 and N861740 of the gene AT5G62150 shows a high resistance against Verticillium , the bars of the mutants are around 0.4 and 0.2, which is lower than the Col-0 which is 1. These data indicate a more resistant mutant.

The mutant N653309 of the gene AT5G65650 shows two different infection bars, both are below the Col-0 baseline which indicates that the data from this mutant not reliable and there should be confirmed if the plants tested are still

segregating for the T-DNA insertion.

The mutants N673884 and N861350 are of the gene AT2G39681 of which N673884 is more resistance and N861350 shows two different bars. The first mutant N673884 shows two bars which are lower than the baseline of Col-0. The second mutant N861350 shows two different bars. The N861350 mutant also requires an additional verification for T-DNA segregation.

In Figure 9 three Arabidopsis mutants that were infected with Verticillium are shown, the mutants are compared with the wild-type (Col-0). These pictures indicate that the biomass quantification is very important to confirm if a plant is more susceptible or resistant than the Col-0 wild-type, because it is difficult to score the susceptibility or resistance based on phenotypic disease symptoms.

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Confirmation of altered gene expression

The gene expression data was tested on the cDNA of sgs2-1 and Col-0 plants harvested at 3, 6 and 9 dpi after Verticillium infection and mock inoculation.

The infection and gene expression assay was repeated once. Figure 10 shows the graph of a few representative target genes. Attachment 1 shows all the assayed genes from the first infection assay and Attachment 2 shows all the genes that were assayed in the second infection.

All three mutants corresponding to the mutated genes in gene AT2G39681, AT5G62150 and AT5G65650 show an up-regulation of the gene transcript at 3 and 6 dpi in sgs2-1, which is in correlation with the transcript RNA sequencing data. Attachment 1 shows that this is consistent for most of the selected genes. In Attachment 2 the data is inconsistent with the first infection assay and the RNA-sequencing transcript data. We have to repeat the expression data analysis to understand this discrepancy; it might be possible that the infection of the second assay was less successful.

Figure 10: Gene expression data after testing at 3, 6 and 9 dpi

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Small RNA confirmation

The small-RNA detection was done on plant tissue of Col-0 and sgs2-1 mutant for 3, 6 and 9 dpi or mock treatment.The small-RNAs are detected by PCR amplification; the method used was published by Varkonyi-Gasic et al. (2007). In brief, the small-RNAs were isolated with the microRNA isolation kit from Sigma. From the isolated RNA cDNA was made by using small RNA specific stem-loop RT primer. On the produced cDNA a PCR was performed. Figure 11 visualizes the small-RNA PCR fragments that were separated a 3% DNA gel (Varkonyi-Gasic, Wu et al. 2007).

During analysis two genes were identified that function as a target of tasiRNA and an origin of small-RNA, this dual function was further analyzed and the validated small-RNAs are shown in Figure 11. In this figure four detected small-RNAs are shown, of which two are tasi-RNAs originating from TAS1A and TAS2 gene. The genomic site of origin of the small-RNA is indicated by the Arabidopsis gene ID behind each panel. The last column in Figure 11 shows the putative gene target of each small-RNA which are also indicated by the Arabidopsis gene ID number.

tasiR1.165 originates from TAS2 gene targets AT1G62910 and AT1G63080 of which the degradation probably leads to the formation of siRNA1.232. Both genes contain pentatricopeptide repeats (PPR) and AT1G63080 also is previously described as a target of ta-siR2140 that is derived from TAS2 (Howell, Fahlgren et al. 2007).

Figure 11: Small-RNA detection

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Discussion

We postulated that the lack of a specific group of tasi-RNAs and their regulated target genes caused the increased susceptibility to Verticillium within the sgs2-1 mutant. In this research we showed that some of the identified tasi-RNAs and their cognate transcript targets are indeed required for full Verticillium colonization. The target genes that were ordered were generated from the list of detected small-RNAs. The data also confirm that the screening for Verticillium colonisation of T-DNA insertion mutant Arabidopsis lines is very powerful.

Although, we should keep in mind that not all predicted homozygous Arabidopsis T-DNA insertion lines are indeed homozygous. The PCR analysis for the T-DNA results show that 26 out of 53 predicted homozygous mutants were indeed homozygous, others were still segregating or even not mutated in the specific gene, therefore further testing is required. This finding was also already published by (Krysan, Young et al. 1999).

To generate homozygous lines, heterozygous lines were seeded again and transplanted, in order to generate more homozygous mutants. The lines that were putative homozygous or heterozygous were seeded again and transplanted, so these could be tested again to obtain more homozygous lines.

We infected 47 mutant lines with Verticillium for the biomass quantification and 25 mutants showed an increased resistance against Verticillium. The plant infection was done twice and both infection assays show increased Verticillium resistance for 22 mutant lines. The pictures of the plants were compared with the quantified biomass data, these show that it is difficult to conclude if plants have increased resistance, but the outcome of the biomass quantification is clear. The second infection assay shows smaller plants and more differences in size between the mutants and Col-0. It is known that Verticillium dahliae can grow better during summer time probably because of this difference between the first and second infection this could explain the larger phenotypic differences after the second infection (Yadeta, 2013).

The gene expression data shows both times that the identified and studies genes were up-regulated within the sgs2-1 mutant when compared to Col-0. These data correlates with some of the small-RNA data where the small-RNA is down-regulated or not detectable on the DNA gel. This phenomenon was meanly visible in the transcript expression data from the first infection assay. The transcript expression of the second infection assay shows less increased transcript accumulation of the selected genes in the sgs2-1 when compared to Col-0. So additional infection assays and expression analysis is required. For one out of the two assays and for multiple genes that were tested we could confirm that an up-regulation for the specific gene transcripts in the sgs2-1 mutant is detectable and this could correlate with the down-regulation of the small-RNA that is targeting this specific gene. In addition, we could validate the importance for Verticillium

colonisation of these specific genes, because mutants that lack the gene are more resistant. This study has also successfully resulted in the validation of a few candidate small-RNAs and their target genes. Therefore in this research we could identify the tasi-RNAs and their cognate accumulated transcript targets that are absent during Verticillium colonization in the sgs2-1 mutant. Indeed we could show that the identified target genes were the key for the basal defence against Verticillium in Col-0.

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Materials and Methods

Selection and ordering of the T-DNA insertion Arabidopsis knock-out lines

The seeds for the knock-out lines were ordered from the European Arabidopsis Stock Centre (NASC): http://arabidopsis.info. The seeds were either homozygous or heterozygous, which was confirmed by PCR. If no homozygous insertion line was available, they were generated from heterozygous lines. These were

transplanted and the offspring was tested for homozygosis.

Root inoculation by Verticillium dahliae

The root inoculation was done with diluted Verticillium dahliae (isolate, JR2). The fungus was first grown on PDA plates for 10-14 days. Spores were harvested in PDB (potato dextrose broth) medium, the amount of isolated spores were counted with a hemocytometer (Burker-Türk). After counting of the harvested spores they were diluted to an amount of 1*106/ml JR2 spores with help of the formula: average number of spores x 16 x 100 x 104. Verticillium infection was done on the roots of 2-week old seedlings of the knock-out lines. The roots were washed twice in water and immersed for 2 minutes in the 1*106/ml JR2 spores. The infected plants were monitored and samples were harvested at 3, 6, 9 or 21 days post infection.

RNA isolation

The RNA isolation was performed using the RNeasy kit of Qiagen according manufactures proceedings on the plants harvested at 3, 6 and 9 days post mock treatment or Verticillium infection. The samples were first grinded in liquid nitrogen and eluted in 80 μl elution buffer.

After the RNA isolation DNase treatment was performed using 1 U/μg RNA RQ RNase-Free DNase (Qiagen). This mixture was incubated at 37 °C for 30 minutes. After this 1 μl of stop solution was added to the mixture, so the DNase was inactivated, the solution was incubated at 65°C for 10 minutes. The amount of RNA was measured with the Nanodrop (Thermo scientific).

cDNA synthesis

cDNA was made using 1 μg of RNA, 1μl (dT) 20 primer, 1 μl dNTPs (10 μm), which had a final volume of 13 μl. This mixture was heated 5 minutes at 65 °C.

After this step a new mixture was made of 4 μl 5x First strand buffer, 1 μl 0.1 m DTT, 1 μl RNaseout/RNase in, 1 μl Superscript 3 RT, which had a final volume of 7 μl. The total volume was 20 μl, this was heated 1 hour at 50 °C and 15 minutes at 70 °C. After the PCR was finished the cDNA was diluted 10 times with dH2O (autoclaved

Milli-Q water).

DNA isolation

The DNA isolation was performed using the DNeasy kit of Qiagen according manufactures proceedings on the plants harvested at 21 days post mock treatment or Verticillium infection. Plants were first grinded in liquid nitrogen. DNA was eluted from the column in 80 μl elution buffer and the concentration was determined with the Nanodrop (Thermo scientific).

Designing of the primers

Primers were designed to detect the gene expression and the T-DNA insertion. The expression primers were designed by the program Primer3 Plus. The position of the primers were verified by the Arabidopsis Information Resource (http://www.arabidopsis.org/servlets/sv), so it was determined that the primers also included the T-DNA insertion. The border primers for the T-DNA insertion are available and the sequence is known, these primers will be combined with the designed primers (Krysan, Young et al. 1999)

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Gene expression detection by qPCR

Gene expression of the selected genes during Verticillium infection vs. mock treatment was detected by qPCR in Col-0 (wild-type) and sgs2-1 (mutant). This detection was performed on 1 μl of the diluted cDNA. In total 12 samples and 8 different primer sets were tested per qPCR run, as a relative loading control the Arabidopsis Actine 2 (Act2) gene was used.

The reaction mixture used for the gene detection contained, 1 μl cDNA sample, 1.25 μl Primer FW (10 μm), 1.25 μl Primer RV (10 μm), 12.5 μl 2x qPCR mix and 5 μl dH2O, the total volume is 25 μl. The qPCR

programme was started with 10 minutes at 95 °C, followed by 40 cycli of 15 seconds denature step at 95 °C and 1 minute anneal step at 60 °C. After the PCR cycles a dissociation step was performed, 15 seconds at 95 °C, 30 seconds at 60 °C and 15 seconds at 95 °C to determine the presence of one specific amplicon.

Verticillium biomass quantification by qPCR

5 ng of isolated DNA was used for each sample. For this qPCR the primers for Ave1 Verticillium and Rubisco (Rub) as a control were used. The mutants were tested in triplet and in total 12 samples could be tested per run.

The reaction mixture for the biomass check contained, 5 μl DNA sample (1 ng/μl), 1.25 μl primer FW (10 μm), 1.25 μl primer RV (10 μm), 12.5 μl 2x qPCR mix and 5 μl dH2O, the total volume was 25 μl. The qPCR run

was started with 10 minutes at 95 °C, followed by 40 cycli of 15 minutes at 95 °C and 1 minute at 60 °C.

Quick genomic DNA Arabidopsis prep

Of the knock-out lines (homozygous or segregating) was 1 leaf cut and a DNA isolation was performed. The leaf was grinded with a drill in a 1.5 ml Eppendorf tube and 400 μl Extraction buffer was added (200 mM Tris (pH7.5), 250 mM NaCl, 25 mM EDTA and 0.5% SDS), the extraction buffer was preheated or the solution with leaf was heated 10 minutes at 65 °C. Samples were centrifuged for 5 minutes at maximum speed. The supernatant was saved and in a concentration of 1:1 iso-propanol was added, the mixture was centrifuged for 5 minutes at maximum speed. The pellet was washed with 500 μl ethanol and centrifuged for 2 minutes at maximum speed. The pellet was dried and dissolved in 100 μl dH2O.

PCR

A PCR was performed on the isolated DNA described above. The PCR was used to detect the T-DNA insertion and the insertion was homozygous if the T-DNA was present in both strands. For each gene a Col-0 control was included. Per sample 2 different primer sets were used, one to detect the gene, the other to detect the T-DNA insertion. In Col-0 samples only the gene primer combination could be detected. Heterozygous lines showed both, an amplicon for the gene primer combination and an amplicon for the T-DNA/gene primer combination. Homozygous insertion lines only gave an amplicon for the T-DNA/gene primer combination.

The PCR mixture contained 1 μl DNA sample, 5 μl 5x Go Taq PCR buffer, 0.25 μl GoTaq, 0.5 μl dNTP’s (10 mM), 1 μl Primer FW (10 μm), 1 μl Primer RV (10 μm), 16.25 μl dH2O, the total volume per reaction was 25 μl.

These were pre-mixtured in two different mixtures, for Col-0 and the rest. The PCR programme was started with 5 minutes at 95 °C, followed by 35 cycli of 1 minute at 95 °C, 1 minute at 58 °C, 2 minutes at 72 °C and a final extention of 5 minutes at 72°C.

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Attachments

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Attachment 2: Gene expression graph of second infection, quantified on 15.04.2013

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Attachment 3: Graph after the biomass quantification, first and second time results are shown

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Attachment 4: small-RNAs and negatively correlated tested target genes

siRNA ID (s) target ID Target description

Chr1_15635171 AT1G09460 Glucan endo-1,3-beta-glucosidase-related

Chr2_16537690 AT1G12775 PPR

Chr1_11550919 AT1G16520 Unknown protein

Chr2_2065682 Chr5_13813897

AT1G29990 PREFOLDIN 6

Chr2_4098433 At1G45474 Pigment binding

Chr3_10293858 AT1G47250 PAF2; threonine-type endopeptidase

Chr2_8577 Chr3_14202548◊

AT1G53070 Lectin family Chr2_3785

Chr3_14197756

AT1G66240 Homolog anti-oxidant 1

Chr1_18599869 AT1G69510 cAMP-regulated phosphoprotein 19-related protein

Chr3_13729852 AT1G72340 eIF-2B family protein

Chr2_3911 Chr3_14197882*

AT2G115520 CRCK3; calmodulin-binding receptor-like cytoplasmic kinase 3

Chr4_1867587 AT2G15840 Pseudogene

Chr3_13632999 AT2G20530 PROHIBITIN 6

Chr3_16142953 AT2G23680 Stress-response protein

Chr4_5178021 Chr5_11639884

AT2G34530 Unknown protein

Chr3_14197882* AT2G37770 Reductase

Chr2_16537648 AT2G39675 TAS1c

Chr2_16539770 AT2G39681 TAS2

Chr3_14202548◊ AT3G06020 Unknown protein

Chr2_4098360 AT3G11780 MD2 domain, Lipid recognition

Chr2_2065693 AT3G16100 GTP binding, ARABIDOPSIS RAB GTPASE HOMOLOG G3C

Chr5_14524394 AT2G26070 Plastid-lipid associated protein

Chr3_1966348● AT4G32130 Carbohydrate binding

Chr2_7583 Chr3_14201554

AT4G37930 Serine-transhydroxy-methyltransferase1

Chr1_11982850 At5G42090 Endomembrane system, integral to membrane

Chr3_1966348● AT5G46780 VQ motif protein

Chr5_12859485 AT5G54610 Ankyrin binding protein

Chr1_23299908 AT5G62150 Peptidoglycan-binding LysM domain protein

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Attachment 5: Tested gene targets for biomass

AGI-code Order number Homozygous/heterozygous Biomass

1st 2nd

AT1G47250 N661036 SALK_098236C Wild-type √

AT1G16520 N665396 SALK_017279C Homozygous after transplanting

AT1G45474 N682226 SALK_036040C Unknown √ √

AT1G45474 N862681 SAIL_682_F10 Wild-type √ √

AT1G69510 N652983 SALK_000600C Homozygous

AT1G72340 N661479 SALK_011277C Homozygous √ √

AT1G66240 N661753 SALK_021013C Unknown √ √

AT1G66240 N678278 SALK_026221C Putative homozygous √ √

AT1G69510 N655672 SALK_083981C Not tested

AT1G29990 N16396 pfd6-1 Not tested

AT2G39681 N861350 SAIL_857_A12 Wild-type √ √

AT2G20530 N682209 SALK_034546C Heterozygous √ √

AT2G37770 N862442 SAIL_71_E07 Wild-type √ √

AT2G11520 N681703 SALK_125879C Homozygous √

AT2G15840 N658930 SALK_136473C Putative homozygous √

AT2G15840 N658875 SALK_118707C Homzoygous √

AT2G27400 N851772 WiscDsLox338F01 Not tested √

At3G61840 N667979 SALK_024950C Not tested √

At3G61755 N669578 SALK_037371C Not tested √ √

AT3G26070 N685926 SALK_051943C Homozygous √

AT3G16100 N665037 SALK_021190C Wild-type √ √

At4G16940 N659276 SALK_032697C Not tested

At4G16870 N673772 SALK_008782C Not tested √ √

At4G16870 N860189 SAIL_45_A05 Not tested √ √

AT4G32130 N671775 SALK_099293C Wild-type √ √

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AT4G37930 N8010 M912 Not tested

AT5G58970 N655650 SALK_080188C Homozygous √

At5G62140 N663702 SALK_113654C Homozygous

At5G18065 N692470 SALK_203943C Homozygous √ √

AT5G39880 N663878 SALK_122791C Homozygous

AT5G39880 N662403 SALK_048931C Heterozygous √

AT5G39880 N859452 SALK_035362 (CD) Not tested

AT5G62150 N681837 SALK_144729C Wild-type; √ √

AT5G62150 N861740 SAIL_1301_C04 Putative homozygous √

AT5G46780 N681089 SALK_035635C Putative homozygous; 1 + 3

The role of RNA silencing in the Verticillium wilt disease

Role of RNA silencing in Verticillium

wilt disease

Role of RNA silencing in Verticillium

wilt disease

Abstract

TABLE OF CONTENTS

Theoretical background

Plant-pathogen model system used for our studies

RNA silencing

Results

Transcript and small RNA isolation of Col-0 and sgs2-1 Arabidopsis plants after Verticillium

infection

Identification of the target genes

Generation of homozygous T-DNA lines

Verticillium colonisation of mutated Arabidopsis lines

Confirmation of altered gene expression

Small RNA confirmation

Discussion

Materials and Methods

Selection and ordering of the T-DNA insertion Arabidopsis knock-out lines

Root inoculation by Verticillium dahliae

RNA isolation

DNA isolation

Designing of the primers

Gene expression detection by qPCR

Verticillium biomass quantification by qPCR

Quick genomic DNA Arabidopsis prep

PCR

Cited literature/ reference

Attachments

Attachment 2: Gene expression graph of second infection, quantified on 15.04.2013

Attachment 3: Graph after the biomass quantification, first and second time results are shown

Attachment 4: small-RNAs and negatively correlated tested target genes

Attachment 5: Tested gene targets for biomass