Microarray analysis of host transcription in response to infection of arabidopsis thaliana protoplasts by coat protein mutants of TCV and HCRSV

MICROARRAY ANALYSIS OF HOST TRANSCRIPTION IN RESPONSE TO INFECTION OF ARABIDOPSIS THALIANA PROTOPLASTS BY COAT PROTEIN MUTANTS OF TCV AND HCRSV LUO QIONG NATIONAL UNIVERISITY OF SINGAPORE 2005 MICROARRAY ANALYSIS OF HOST TRANSCRIPTION IN RESPONSE TO INFECTION OF ARABIDOPSIS THALIANA PROTOPLASTS BY COAT PROTEIN MUTANTS OF TCV AND HCRSV LUO QIONG (M Sc., CAAS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPROE 2005 Acknowledgements I would like to thank my supervisor A/P Wong Sek Man for his guidance, encouragement and help Dr Wong put in great efforts in designing the project, solving problems in the research, and helping me with my thesis Without his guidance and help, what was done in the past a few years would have been impossible I would like to thank A/P Peng Jinrong from IMCB for his advice and assistance My thanks also go to Dr Jin-Hua Han, Mr Chong Ping Lee, Ms Soo Hui Meng and Mr Wu Wei for their professional support I am also very grateful to Dr Li Weimin and Meng Chunying for their invaluable suggestions and help And I want to say thank you to former and current lab mates Dr Wang Haihe, Dr Srinivasan KG, Dr Lee Kian-Chung, Lena, Chinchin, Shishu, Wee Su and Dai Liang Their friendships, encouragements and help have made my work and study easier and more enjoyable I would also acknowledge the financial support of National University of Singapore and emotional support of my family and friends through this special and important period of my life I Table of Contents Acknowledgements………………………………………………………I Table of Contents……………………………………………………… II Summary………………………………………………………… … VII List of Tables…………………………………………………………….i List of Figures……………………………………………………………i List of Symbols………………………………………………………….iv Chapter Introduction 1.1 TCV and HCRSV……………………………………………………1 1.1.1 Similar genome organization.…………………………………1 1.1.2 Different host ranges and symptoms ………………… 1.1.3 Research of interaction with mutation……………………… 1.2 Host-virus interaction involving CP…………………………………5 1.2.1 Coat protein as an important component of virus…………… 1.2.2 TCV and HCRSV interaction with host……………………….7 1.3 Investigation of Arabidopsis-pathogen interaction………………….8 1.3.1 Arabidopsis thaliana genome sequencing project…………….8 1.3.2 Microarray analysis method………………………………… 1.3.3 Microarray analyses on Arabidopsis response to viruses……10 1.4 Objectives………………………………………………………… 11 II Chapter Materials and methods 2.1 Biological materials………………………………………………12 2.1.1 Bacterial strains……………………………………………12 2.1.2 Plasmid vectors ………………………………………… 12 2.1.3 Culture medium………………………………………… 12 2.1.4 Animal for production of antisera……………………… 13 2.1.5 Plant materials ……………………………………………13 2.1.6 Microarray of Arabidopsis……………………………… 13 2.2 Preparation and transformation of competent cells……………….13 2.3 Overlapping polymerase chain reaction (PCR) ………………… 15 2.4 In vitro transcription and purification of transcripts…………… 16 2.5 Generation of DIG-labeled RNA probes …………………………17 2.6 Screening of transformants……………………………………….17 2.6.1 Small scale purification of plasmid DNA……………… 17 2.6.2 Restriction digestion …………………………………… 19 2.6.3 Automatic DNA sequencing …………………………… 19 2.7 Inoculation of plants………………………………………………20 2.8 Northern blot analyses ……………………………………………20 2.8.1 Separating RNA on denaturing gel…… ……………… 20 2.8.2 Transfer and blotting …………………………………… 20 2.9 Western blot analyses…………………………………………… 21 Chapter Expression of TCV CP and HCRSV CP and production of antibodies 3.1 Introduction………………………………………………………24 III 3.2 Materials and methods……………………………………………26 3.2.1 Construction of pET32-H-TCVCP and pET32-H-HCRSVCP ………………………………… 26 3.2.2 Expression and purification of the CPs………………… 26 3.2.2.1 Purification of the TCV CP…………………….27 3.2.2.2 Purification of the HCRSV CP……………… 28 3.2.3 Immunization and tests of sensitivity and specificity…….31 3.3 Results and discussion………………………………………… 31 3.3.1 Expression of TCV and HCRSV CPs…………………… 31 3.3.2 Test of antibody against TCV CP and HCRSV CP……….32 Chapter Construction of mutants TCV-CPHCRSV and HCRSV-CPTCV 4.1 Introduction ……………………………………………………….39 4.2 Materials and methods…………………………………………….40 4.2.1 Construction of TCV-CPHCRSV ……………………………40 4.2.2 Construction of HCRSV-CPTCV………………………… 41 4.2.3 Replication of mutant in vitro…………………………… 42 4.3 Results and discussion…………………………………………….42 Chapter Replication of viruses and expression of CP genes in protoplasts and whole plants 5.1 Introduction……………………………………………………… 47 5.2 Materials and methods.……………………………………………48 5.2.1 Arabidopsis protoplast isolation and transfection……… 48 IV 5.2.2 Hibiscus protoplasts isolation and transfection……………48 5.2.3 Replication and expression CP genes in protoplasts………50 5.2.4 Replication and expression CP genes in plants.………… 52 5.3 Results and discussion…………………………………………… 53 5.3.1 Replication and expression of TCV, TCV-CPHCRSV, HCRSV, HCRSV-CPTCV in Arabidopsis and Hibiscus cannabinus protoplasts………………………… 53 5.3.2 No complementation of HCRSV and TCV, or TCV-CPHCRSV and HCRSV-CPTCV in whole plant……… 54 Chapter Arabidopsis microarray analyses of TCV, HCRSV, TCV-CPHCRSV and HCRSV-CPTCV 6.1 Introduction……………………………………………………….61 6.2 Materials and methods…………………………………………….63 6.3 6.2.1 RNA sample preparation………………………………….63 6.2.2 Microarray analysis……………………………………….64 6.2.2.1 First-strand cDNA synthesis…………………….64 6.2.2.2 Second-strand cDNA synthesis………………….65 6.2.2.3 Clean up of double-stranded cDNA…………… 65 6.2.2.4 Synthesis of biotin-labeled cRNA……………….66 6.2.2.5 Clean up and quantification of IVT products……66 6.2.2.6 Fragmentation of cRNA ……………………… 67 6.2.2.7 Eukaryotic hybridization……………………… 67 6.2.2.8 Array wash and stain…………………………….69 Results …………………………………………………………….70 6.3.1 cRNA sample preparation…………………………………70 V 6.3.2 Expression profiles of protoplast samples…………………72 6.3.2.1 Consistency analysis and preanalysis of data…….72 6.3.2.2 K-Means clustering………………………………73 6.3.2.3 Two-way ANOVA………………………………84 6.3.2.4 Pair-wise comparison ……………………… 89 6.4 Conclusion and discussion……………………………………… 90 References ……………………………………………………………93 Appendix …………………………………………………………… 101 VI Summary With similar genome organization and replication strategies but with different host ranges, Turnip crinkle virus (TCV) and Hibiscus chlorotic ringspot virus (HCRSV) were chosen to construct coat protein (CP) mutants in this research And the two mutants of TCV-CPHCRSV and HCRSV-CPTCV were made by exchanging CP reciprocally Because viral CPs play important roles in viral infection and replication, by comparing the transcript profiles of Arabidopsis protoplasts infected with wild-type viruses and CP mutants, Arabidopsis genes interacting with virus would be screened out, especially those interacting with these two CP genes Then the infectivity of TCV-CPHCRSV and HCRSV-CPTCV in both Arabidopsis and Hibiscus protoplasts was checked It was shown that in Arabidopsis protoplasts both mutants and their wild-type viruses could replicate and express their CPs, while in Hibiscus protoplasts only TCV, HCRSV and HCRSV-CPTCV could replicate and express their CPs This result implied that some processes were disturbed in the hostmutant interaction and some genes should be involved in this process Arabidopsis genomic microarray analysis was carried out to detect different global gene expression profiles resulting from different interactions with the host genes, which were indicated by the different expression levels of the viruses and mutants in Arabidospsis and Hibiscus protoplasts And several data interpretation methods such as K-means clustering and two-way ANOVA were used to screen for the interesting genes from all the 22751 genes on the Arabidopsis chips VII Through K-Means clustering, it was found that the expression levels of many genes were depressed or activated by the host-virus interaction, and many of these genes were involved in transcription regulation, signal transduction, defense/stress response, and protein degradation machinery and so on Moreover, many functionally unknown genes were also grouped in the clustering, which were considered to be putative genes that may play similar roles in the host-virus interaction And by two-way ANOVA, three groups of genes were shown in Venn Diagram, where each group of genes were differentially expressed under the influence of interaction of host-CP, host-viral backbone (viral genes other than CP genes), hostinteraction of CP and viral backbone, respectively In short, groups of genes with known or unknown functions were picked out as promising candidate genes which are likely to play important roles in plant-virus interaction Starting with identification of Arabidopsis genes that interact with CPs and other viral genes from TCV and HCRSV, exact roles played by host genes in response to viral infection and the mechanisms of host-virus interaction may be ultimately made clear VIII Table 6.2 Genes whose expression levels were influenced by interaction between host and virus CPs Functional class Unclassified Unknown Unknown Transporters Interesting miscellaneous Interesting miscellaneous Interesting miscellaneous Unclassified Unclassified Unknown Kinase Transcript ID_Affymetrix Gene Title_Affymetrix pseudogene, similar At3g07030 OSJNBa0042L16.13 At5g45480 expressed protein At5g51240 At5g09400 potassium transporter family protein At2g46060 transmembrane protein-related At5g59750 riboflavin biosynthesis protein, putative At3g03420 At2g19750 At5g09670 At5g02710 At2g33840 At3g47940 At5g40500 Ku70-binding family protein 40S ribosomal protein S30 (RPS30A) loricrin-related expressed protein 3-phosphoinositide-dependent protein kinase, putative tRNA synthetase class I (W and Y) family protein DNAJ heat shock protein, putative expressed protein At5g41150 At2g36485 repair endonuclease (RAD1) (UVH1) expressed protein At4g16120 At2g33180 phytochelatin synthetase-related expressed protein UDP-glucoronosyl/UDP-glucosyl transferase family protein expressed protein At5g04510 Interesting miscellaneous Heat shock protein Unknown Interesting miscellaneous Unknown Interesting miscellaneous Unknown Defense related Unknown Interesting miscellaneous Interesting miscellaneous Interesting miscellaneous to At5g05870 At1g12120 At2g13970 At4g36360 At1g47290 Mutator-like transposase family beta-galactosidase, putative/lactase, putative 3-beta hydroxysteroid dehydrogenase/isomerase family protein 87 Table 6.3 Genes whose expression levels were influenced by interaction of host-interaction between viral backbone and CP Functional class Interesting miscellaneous Defense related Defense related Interesting miscellaneous Defense related Unknown Interesting miscellaneous Hydrolase Protease Unclassified Defense related Transporter Unknown Unknown Unclassified Heat shock protein Unclassified Interesting miscellaneous Unknown Unknown Transporter Heat shock protein Interesting miscellaneous Unknown Transcript ID_Affymetrix Gene Title_Affymetrix transducin family protein/WD-40 repeat At2g37160 family protein At1g68520 At3g53160 At2g17430 At5g24970 At5g07950 zinc finger family protein UDP-glucoronosyl/UDP-glucosyl transferase family protein seven transmembrane MLO family protein / MLO-like protein At3g28030 At5g59250 At5g43180 At1g49170 At5g09670 ABC1 family protein expressed protein tRNA-nucleotidyltransferase, putative / tRNA adenylyltransferase, putative glycosyl hydrolase family protein protease inhibitor/seed storage/lipid transfer protein (LTP) family protein U-box domain-containing protein UV hypersensitive protein (UVH3) / DNArepair protein, putative sugar transporter family protein expressed protein expressed protein loricrin-related At3g47940 At5g56940 DNAJ heat shock protein, putative ribosomal protein S16 family protein At3g28850 At4g24200 At3g63210 At3g59140 glutaredoxin family protein expressed protein expressed protein ABC transporter family protein At5g23590 DNAJ heat shock protein At5g56600 At1g30135 profilin (PRO5) (PRF3) expressed protein At1g22660 At2g44460 At4g12490 At3g52450 88 6.3.2.4 Pairwise comparison of samples In two-way ANOVA, 1.5 times change in gene expression level was set as the threshold to retain potentially useful data for further analysis Even though this criterion was not very stringent, it was still possible that some useful data were neglected because only the difference caused by interaction between host and viral backbone or CP was considered So it was helpful to use another method by consideration of a large range of genes in the analysis Pairwise comparison was adopted for this purpose, which compared any pair of samples instead of just judged the change of expression level caused by interaction of host-viral backbone or host-CP as two-way ANOVA did At the same time, a more stringent selection criteron of at least times was used to get a manageable amount of differentially expressed genes As a result, a few hundred of genes were obtained (data not shown), and then following the method of a previous study (Marathe et al, 2004) they were divided into ten functional classes (Table 6.4) Table 6.4 Ten classes of genes were differentially expressed upon viral infection of Arabidopsis protoplasts 10 Functional classes Defense related Kinases/phosphatases Transcriptional regulators Protein degradation machinery/proteases Heat shock proteins Lipases/hydrolases ROI related Transporters Unknown and unclassified Interesting miscellaneous 89 6.4 Conclusion and discussion In this study we obtained CP mutants of TCV-CPHCRSV and HCRSV-CPTCV and found that both of them retained infectivity (but weaker than the wild-type viruses’) in Arabidopsis protoplasts, and HCRSV-CPTCV retained infectivity while TCV-CPHCRSV lost its infectivity in Hibiscus protoplasts This fact suggested that some processes were disturbed in the host-mutant interaction and some genes may be involved in this process Based on these resutlst, we propose a hypothesis as stated below Host cells respond differently to infection of wide type virus TCV, HCRSV, and their CP mutants TCV-CPHCRSV and HCRSV-CPTCV, leading to different RNA profiles of Arabidopsis protoplasts in the microarray analysis And while in the interaction of host and virus, the host response will then be strengthened or weakened in CP mutantsinfected cells compared with those infected with wide-type due to different gene-gene interaction caused by the chimeric viruses By comparison of the profiles through complicated data interpretation, we are able to identify candidate genes specifically responding to CP gene, or other viral genes So we intended to find the putative genes that function in the process of interaction between host and viruses, and microarray analysis was chosen to meet this aim And for the data we obtained, K-means clustering, two-way ANOVA and pairwise comparison were selected to search for the interesting genes from all the genes on the Arabidopsis chips Through K-Means clustering, we found some genes whose expression levels were influenced by the host-virus interaction and these genes were mainly involved in 90 transcription regulation, signal transduction, protein synthesis and modulation, and defense/stress reponse Moreover, many functionally unknown genes were also grouped in the clustering, which were considered to be putative genes that may play some roles in the host-virus interaction All the genes mentioned above responded to viral infection in a virus-general or viral-specific way, such as genes specifically upregulated by TCV or downregulated by HCRSV CP gene These results strongly support our hypothesis By two-way ANOVA, we identified three groups of genes which were differentially expressed under the influence of interaction of host-CP, host-vrial backbone, host-interaction of CP and vrial backbone This result demonstrated that the CP alone or the interaction between CP and other viral genes had great influence on the host-virus interaction, and further study on these genes will throw light on the mechanisms of host-virus interaction And pairwise comparison produced listes of genes which were divied into nine functional classes, including defense related, transcription regulation, heat shock protein and protein degradation machinery and others, which were previously reported as factors involved in host response to viral infection (Marathe et al, 2004) This method can act as a supplementary method to search for differentially expressed genes that might be neglected in other cases In summary, groups of genes with known or unknown functions were picked out as promising candidate genes which are likely to play important roles in plant-virus interaciton Starting with identification of Arabidopsis genes that interact with CPs 91 from TCV and HCRSV and other viral genes that play roles in host response to viral infection, we hope 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TCVpri4 HCPmuta1 HCPmuta2 HCRSVpri1 HCRSVpri2 Sequence 5-CGGAATTCCCTACAACTCTCTAAGCG-3 5-CTTCTGCAGCATTTCCAGTGTTGATGCTTA-3 5-GCCTGTAGGAACTAGTACGGTAATAGTGTA-3 5-GGACTAGTTTTCCAGTCTAATGCCCGCA-3 5-TCAACACTGGAAATGCTGCAGAAGAATGAC-3 5-ACTATTACCGTACTAGTTCCTACAGGCCCA-3 5'-CATGCCATGGACATTGAGCTTGAG-3' 5'-TTGCAAGCACTCAGAATTTAGGTG TGTATTGTATGGGCCTCCGTG-3' HCRSVpri3 5'-CACGGAGGCCCATACAATACACAC CTAAATTCTGAGTGCTTGCAA-3' 10 HCRSVpri4 5'-TCCCCGCGGTGGCGGCCGCTCTAG-3' 11 TCP1 5'-CATGCCATGGCTATGGAAAATGATCCTAGAGTC-3' 12 TCP2 5'-CCGCTCGAGTTAAATTCTGAGTGCTTGC-3' 101 [...]... to show how to construct HCRSV- CPTCV Figure 4.3 TCV- CPHCRSV and HCRSV- CPTCV produced RNA by in vitro transcription i Figure 5.1 TCV, TCV- CPHCRSV, HCRSV and HCRSV- CPTCV could replicate in Arabidopsis protoplasts Figure 5.2 TCV, HCRSV and HCRSV- CPTCV could replicate in Hibiscus protoplasts Figure 5.3 TCV, TCV- CPHCRSV, HCRSV and HCRSV- CPTCV could express CP in Arabidopsis protoplasts Figure 5.4 TCV, HCRSV. .. were motivated to use it to provide some insights into the global response of Arabidopsis protoplasts to infection of these two viruses and their mutants 1.4 Objectives 1 To construct two virus mutants: HCRSV- CPTCV and TCV- CPHCRSV 2 To transfect A thaliana and H cannabinus protoplasts with TCV, HCRSV, TCV- CPHCRSV and HCRSV- CPTCV and to detect their replication and CP expression 3 To investigate if... would allow HCRSV- CPTCV and TCVCPHCRSV to systemically infect A thaliana, N benthamiana and H cannabinus, respectively and to check virus complementation between TCV and HCRSV or between TCV- CPHCRSV and HCRSV- CPTCV 4 To compare the gene expression profiles of Arabidopsis protoplasts transfected with TCV, HCRSV and their mutants using Arabidopsis oligonucleotide microarray 11 Chapter 2 Materials and methods... and Hibiscus chlorotic ringspot virus (HCRSV) to construct coat protein (CP) mutants And then we tested viral mutants by checking their infectivities Finally, to study the mechanism of interaction, we used Arabidopsis microarray to find out candidate genes that may be involved in host- virus interaction 1.1 TCV and HCRSV 1.1.1 Similar genome organization TCV and HCRSV, as members of the genus Carmovirus,... production of antiserum New Zealand rabbits were used in production of antiserum against bacterial expressed TCV and HCRSV CPs 2.1.5 Plant materials For testing of systemic infection of TCV, HCRSV, TCV- CPHCRSV and HCRSVCPTCV, Arabidopsis thaliana, Hibiscus cannabinus and Nicotiana benthamiana were used Both A thaliana and H cannabinus were used as the starting material for isolating protoplasts All plants of. .. conformation, thereby affecting interactions between CP and plant hosts (Lin and Heaton, 1999) Moreover, Nterminus of the TCV CP was shown to be involved in eliciting resistant responses in Di-17 Arabidopsis, suggesting CP was the avirulence factor recognized by the resistant host (Zhao et al, 2000) On the other hand, some plant factors were found to interact with TCV (Ren et al, 2000; Lin and Heaton, 2001;... with water, HCRSV- CPTCV, TCV- CPHCRSV, HCRSV and TCV (from lanes 1 to 5) Figure 6.2 Scatter plot of data from negative controls in two microarray analysis duplicates indicated the experiment was reproducible Figure 6.3 Candidate genes, whose expression levels were lower in samples HCRSV and TCV- CPHCRSV compared with negative control, TCV and HCRSV- CPTCV, may be involved in interaction of host- HCRSV CP... 3.4 TCV CP antibody titer 1 X 106 against virion antibody at 1 X 105 in detection of TCV in infected N benthamiana Figure 3.5 HCRSV CP antibody titer 1 X 105 against virion antibody at 1 X 104 in detection of HCRSV in infected H cannabinus Figure 3.6 Test of specificity of TCV CP (A) and HCRSV CP (B) antibodies in infected H cannabinus protoplasts Figure 4.1 Diagram to show how to construct TCV- CPHCRSV... Figure 5.4 TCV, HCRSV and HCRSV- CPTCV could express CP in H.cannabinus protoplasts Figure 6.1 cRNA sample preparation from total RNA of Arabidopsis for microarray analysis Panel A: cDNA derived from total RNA samples of Arabidopsis protoplasts; Panel B: cRNA generated from cDNA; Panel C: Biotin labeling of cRNA derived from RNA samples of Arabidopsis protoplasts In three panels, protoplasts were transfected... nucleic acid, movement between cells and organs and travel from infected to uninfected plants via biological vectors, induction of host defense machinery and suppression of host silencing (Callaway et al, 2001; Lu et al, 2004) In the following parts, studies on CP interaction with the host such as suppression of host silencing, eliciting symptoms, modifying symptom and enabling viral movement will be briefly .. .MICROARRAY ANALYSIS OF HOST TRANSCRIPTION IN RESPONSE TO INFECTION OF ARABIDOPSIS THALIANA PROTOPLASTS BY COAT PROTEIN MUTANTS OF TCV AND HCRSV LUO QIONG (M Sc., CAAS)... virus mutants: HCRSV- CPTCV and TCV- CPHCRSV To transfect A thaliana and H cannabinus protoplasts with TCV, HCRSV, TCV- CPHCRSV and HCRSV- CPTCV and to detect their replication and CP expression To investigate... TCV- CPHCRSV, HCRSV and HCRSV- CPTCV could replicate in Arabidopsis protoplasts Figure 5.2 TCV, HCRSV and HCRSV- CPTCV could replicate in Hibiscus protoplasts Figure 5.3 TCV, TCV- CPHCRSV, HCRSV and HCRSV- CPTCV