Progress 01/01/14 to 12/31/17
Outputs
Target Audience:Weed scientists, basic biologists interested in parasitic plants, applied scientists interested in crop productivity Changes/Problems:Since the writing of this proposal, gene knockouts using CRISPR technology took a leap forward in tomato. We chose this method for targeted gene knockouts in the host. We also developed an in-vitro haustorium system to monitor the number of haustoria on plates.We also showed that Cuscuta can parasatize tomato hairy roots. What opportunities for training and professional development has the project provided?One graduate student has been able to present her work at several conferences: the American Society of Plant Biologists (ASPB) Plant Biology conference 2017 in Honolulu, Hawaii, UC Davis Plant Biology Colloquium, UC Davis Plant Biology Retreat, Solanaceae Annual Conference 2016 in Davis, CA. One post-doc attended the2016 IS-MPMI XVII Congress in Portland, OR and presented work at the Solanaceae Annual Conference 2016 in Davis, CA. This post-doc has since become a staff Scientist at Zymergen INC, Emeryville, CA. One undergraduate student was trained to do histology and molecular biology including CRISPR cloning. This student presented a poster at the 2017 UC Davis Undergraduate Research Conference. This student has since been elevated to a Jr Specialist position. How have the results been disseminated to communities of interest?This project hosted one high school student during the summer. Besides learning basic laboratory techniques, the student was encouraged to attend lab meetings and presentations so that she is able to see how projects manifest with collaboration and input from everyone in lab. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACT Parasitic flowering plants directly attach to host plants using specialized organs known as haustoria, which function as physiological bridges to extract nutrients and water from their hosts. Cuscuta species (dodders) are agriculturally destructive flowering stem parasitic plants and may lead to a 50-72% reduction in yield in tomato alone. Development of effective and sound management systems to control parasitic weeds is limited because the molecular, genetic, and biochemical factors that govern the interaction of parasitic weeds with their hosts are still unknown. We produced extensive transcriptional data in different parasite tissues including haustoria (Ranjan et al, 2014). Analysis of gene expression dynamics during dodder development and parasitism identified key gene categories involved in the process of plant parasitism. Laser-capture microdissection (LCM) coupled with a time-course experiment identified gene expression changes in specific cells in the tomato shoot that surround the penetrating dodder haustoria. This led us to identify key tomato defense response genes, which we tested through RNAi lines in tomato and Arabidopsis subjected to Cuscuta infection. While most tomato cultivars can be parasitized by C. pentagona, we obtained two hybrid cultivars 9492 and 9553 (H9492 and H9553), which exhibited resistance to dodders. Our results indicate that this resistance relies on a local lignification in the stem cortex upon dodder attachment, creating a barrier to haustorium penetration and inability of the dodder strand to survive on the resistant cultivars. Our results will help develop a parasite-resistant system in crops to reduce economic losses in agriculture. Accomplishments of Goals I. Assemble and analyze the complete transcriptome of dodder using multiple developmental and host-colonization stages. Our de-novo assembled and annotated transcriptome of the obligate plant stem parasite Cuscuta pentagona was used to analyze gene expression dynamics during dodder development and parasitism. Host plant infection is accompanied with increased expression of parasite genes underlying transport and transporter categories, response to stress and stimuli, as well as genes encoding enzymes involved in cell wall modifications. Based on differential and coexpression gene analysis we prioritized a dodder LBD-like transcriptional regulator (Lateral Organ Boundaries Domain gene 25) for initial functional analysis. A fragment from the target gene cloned into RNAi vectors under the phloem-specific SUC2 promoters was used to generate tomato plants for cross-species silencing. We evaluated the growth of C. pentagona on the RNAi inducing tomato host and saw reduced fitness and less haustorial development of the parasite, suggesting that CpLBD25 may be a central regulator of haustoria development. II. Analyze parasite and host structure and gene expression during parasitism The Parasite: Using differential gene expression, coexpression network analysis and predictions of secreted proteins we identified a set of genes specific to the infecting tissue that play a central role in the gene expression network regulating parasitism. Fourteen candidate genes were subjected to functional analysis by host-induced gene silencing in the parasitic weed. Dodder showed impaired growth when a WRKY6-like transcription factor (CpWRKY6) or a HB-7 transcription factor (CpHB7) was targeted for silencing, and improved when a pectin methylesterase inhibitor (CpPMEI) gene was targeted for silencing. The Host: We used the LCM collected RNA-seq data from the host side and complemented it with an additional time course RNA-seq of early attachment stages (days 1 to 4 after attachment). Most expression changes in the host occur at the early stage of attachment. We performed promoter motif enrichment analysis on the promoters of DE genes at 4 DPA and found significant enrichment of binding sites that are putatively regulated by ethylene response factors (ERFs), WRKY and BEL transcription factors. Furthermore, several WRKY and ERF transcripts were significantly upregulated at 4 DPA. III. Prospect for and downregulate key host/parasite genes by targeted RNAi in the host. Host-Parasite interaction in Susceptible tomato - the Host: Since the writing of this proposal we chose CRISPR for targeted gene knockouts (KO) in the host. Based on early gene expression changes in tomato after dodder attachment we identified guide RNAs to target our top 3 candidates, a homolog of PR1, a WRKY16, and an NBS-LRR like gene for functional analysis using CRISPR and generated transgenics. Our preliminary results suggest that the PR1 and NBS-LRR CRISPR lines enhance the ability of dodder to parasitize tomato. The WRKY CRISPR lines form haustoria that are not able to fully penetrate to the host vascular system. Host-Parasite interaction in Resistant tomato - the Host: To date, there are very few identified resistance mechanisms contributing to parasitic weed resistance. We obtained hybrid cultivars that were reported as resistant (H9492 and H9553) to C. pentagona compared to other related susceptible hybrids (H1706 and H9775). We challenged the resistant and susceptible tomato cultivars to C. pentagona and constructed RNA-seq libraries from the 4 genotypes. The transcriptional differences in response to dodder involved only 36 genes scattered across the chromosomes. The genes include several lignin biosynthetic genes that were upregulated in resistant cultivars. We determined that the resistance mechanism involves local lignification in the stem cortex upon dodder attachment to the resistant lines. This lignification created a barrier to haustorium penetration causing the dodder strand to fall off the resistant cultivar. We found an AP2, a MYB, and a CC-NBS-LRR gene involved in regulation of local lignification of stems. Host-Parasite interaction in Susceptible tomato - the Parasite: Host-based RNA interference has been successfully used as a "Trojan-horse" mechanism to suppress the development of dodder. Using a transcriptomic approach, we identified haustoria specific novel genes as putative regulators for parasite development using our bioinformatics pipeline. We generated tomato and Arabidopsis RNAi lines targeting Cuscuta transcripts. Tomato was used for the 4 top candidates (CpLBD25, CpERF1, CpHB7 and CpPMEI), and Arabidopsis was chosen as a fast approach to screen a larger number of other targets. To evaluate the effect of cross-species RNAi on dodder growth, transgenic RNAi T1 (tomato) and T2 (Arabidopsis) lines were infected and sampled for structural and molecular changes in parasite development. We see that dodder development is affected in Arabidopsis CpPMEI (a pectin methylesterase inhibitor gene), CpHB7 (a drought responsive homeodomain TF gene), and CpWRKY6 RNAi lines. While dodder growth on CpWRKY6 RNAi hosts is hampered, it is somewhat improved on CpPMEI RNAi host plants. IV. Analyze differential gene expression between the different Solanaceae species parasitized by dodder. S. lycopersicum, N. benthamiana and S. habrochaites respond differently to infestation of C. pentagona. Host gene expression from tomato and tobacco was analyzed and reported (Ranjan et al, 2014). To shed light on natural resistant mechanism to parasitic plants in S. habrochaites (HAB) we conducted a screening of tomato HAB introgression lines and informative lines will be assayed further. In addition, we have reason to believe that the Galapagos tomatoes, including Solanum galapagense, could be an important source of resistance to dodder infestations and we are analyzing whole genome sequencing data to identify such genes. References: Ranjan, A., Ichihashi, Y., Farhi, M., Zumstein, K., Townsley, B., David-Schwartz, R., and Sinha, N. (2014) De Novo Assembly and Characterization of the Transcriptome of the Parasitic Weed Dodder Identifies Genes Associated with Plant Parasitism. Plant Physiology 166 (3): 1186-1199.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Jhu, M.Y., Farhi M., Philbrook R., Ranjan A., Ichihashi Y., Khuu K., Liew V., Sinha N. (2017). Developing a parasite-resistant system in tomatoes to control infestations of Cuscuta pentagona. 2017 UC Davis Plant Biology Retreat, UC Davis Genome Center, California,USA (conference poster).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Jhu, M.Y., Farhi M., Ranjan A., Ichihashi Y., Philbrook R., Khuu K., Sinha N. (2016). Developing a parasite-resistant system in tomatoes to control infestations of Cuscuta pentagona. 2016 UC Davis Plant Biology Retreat, Marconi Conference Center, California, USA (conference poster).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Sinha, NR (2017) The interaction between Cuscuta and its host, tomato. ICPDB, NISER, Bhubanehswar, India (invited talk)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Jhu, M.Y., Sinha N. (2017). Developing a parasite-resistant system in tomatoes to control infestations of Cuscuta pentagona. Presentation Competition in 2017 UC Davis Plant Biology Colloquium, UC Davis, CA, USA (oral presentation)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Jhu, M.Y., Farhi M., Philbrook R., Ranjan A., Ichihashi Y., Khuu K., Liew V., Sinha N. (2017). Developing a parasite-resistant system in tomatoes to control infestations of Cuscuta pentagona. American Society of Plant Biologists (ASPB) Plant Biology conference 2017 in Honolulu, Hawaii, USA (conference poster).
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Progress 01/01/16 to 12/31/16
Outputs
Target Audience:Weed scientists, basic biologists interested in parasitic plants, applied scientists interested in crop productivity Changes/Problems:Genetic transformation of tomato cultivar Heinz 1706, for which the genome sequence is available, proved to be slow. We therefore chose to validate the expression patterns of chosen candidate genes in a less recalcitrant cultivar - M82. To keep the time track we perform further work in M82 genetic background. No additional and/or special reports are required. What opportunities for training and professional development has the project provided?Two graduate and two undergraduate students were trained in molecular biology, genomics and bioinformatics during this reporting year. One post-doc attended MPMI and SolGenomics conferences. (Moran) How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We generated tomato and Arabidopsis RNAi lines targeting Cuscuta transcripts that we identified using our bioinformatics pipeline as putative regulators for parasite development. Tomato was used for the 4 top candidates (CpLBD25, CpERF1, CpHB7 and CpPME1), and Arabidopsis was chosen as a fast approach to screen a larger number targets. To evaluate the effect of cross-species RNAi on dodder growth, transgenic RNAi T1 (tomato) and T2 (Arabidopsis) lines were infected in a synchronized manner at an early age with dodder strands, and sampled for structural and molecular changes in parasite development. Our screen showed that dodder development is affected in Arabidopsis CpCLV3 (a CLE gene) and CpWRKY6 RNAi lines. Additionally, we identified several CpLBD25 tomato RNAi T2 lines that show expression of the hairpin and have reduced dodder growth in the greenhouse and suppress haustoria development, as measured in an in-vitro haustorial induction assay. To study the host response to parasitism by dodder we chose a short list of candidate genes that we found to be highly induced in tomato upon Cuscuta attachment. We first validated the expression of the selected genes using qRT-PCR in the tomato M82 cultivar which is more amenable to genetic transformation compared to Heinz 1706. For functional analysis using CRISPR/Cas9 targeted gene knockout we identified guide RNAs to target the first 3 candidates, including a homolog of PR1, WRKY71-type, and an NBS-LRR like gene. Next, we generated transgenic CRISPR M82 plants that show mutation in these targets and we'llbe evaluating Cuscuta growth on them once T1 plants are ready. We have obtained and tested commercial hybrid cultivars that were reported as resistant to C. pentagona compared to other related susceptible hybrids. We then analyzed the transcriptomic changes that are specific to resistance with the aim of identifying the underlying mechanism. This RNA-seq analysis suggested changes related to cell wall fortification that is unique to the resistant lines, which we corroborated with a histological examination. Furthermore, by resequencing of these cultivars we will soon be able to identify the underlying genetic loci that govern this resistance to the parasitic plant.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Farhi, M. Presentation: Wolf peach vs. strangleweed: untangling the plant-parasitic plant interaction between tomato and Cuscuta. Plant Biology Seminar Series, Davis, CA, Aug. 2016
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Farhi, M, Jhu, M-Y, Philbrook, R, Khuu, K, Sinha, NR. Poster presentation: Analysis of the intimate interaction between tomato and the parasitic plant Cuscuta pentagona. Solanaceae Annual Conference, Davis, CA, Sep. 2016
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Jhu, M-Y, Farhi, M, Philbrook, R, Sinha, NR. Poster presentation: Developing a parasite-resistant system in tomato to control infestations Cuscuta pentagona. Annual Plant Biology Retreat, Marconi Conference Center, CA, Oct. 2016
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Farhi, M. Presentation: Wolf peach vs. strangleweed: the wolf wins! Analyzing tomato resistance to the parasitic plant Cuscuta. Annual Plant Biology Retreat, Marconi Conference Center, CA, Oct. 2016
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Progress 01/01/15 to 12/31/15
Outputs
Target Audience:Weed scientists, basic biologists interested in parasitic plants, applied scientists interested in crop productivity Changes/Problems:Genetic transformation of tomato cultivar Heinz 1706, for which the genome sequence is available, proved to be slow. We therefore chose to validate the expression patterns of chosen candidate genes in a less recalcitrant cultivar - M82. To keep the time track we perform further work in M82 genetic background. No additional and/or special reports are required. What opportunities for training and professional development has the project provided?One post-doctoral student and one graduate student were trained in molecular biology, genomics and bioinformatics during this reporting year. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period we plan to include validation of gene expression changes in the interaction of dodder and tomato. We expect to have validated and in-depth analysis of the interaction of dodder grown on RNAi tomato suppressing the expression of LBD25 and additional genes. We expect that the screening of candidate parasite genes using arabidopsis host induced gene silencing will identify more targets in the parasite for further analysis. Furthermore, we anticipate that we will have initial analysis of tomato plants mutated at key genes that are involved in regulating the defence response to the parasite. Additionally, we will advance the comparative transcriptomic analysis of the response to dodder across the 3 Solanaceae species. ?
Impacts
What was accomplished under these goals?
The molecular mechanisms underlying plant parasitism and host responses are poorly understood and likely involve plant-plant crosstalk and numerous genes/gene-regulatory changes. In order to investigate the process of parasitism, RNAs from different stages, i.e. seed, seedling, vegetative strand, prehaustoria, haustoria, and flowers, were used to de-novo assemble and annotate the transcriptome of the obligate plant stem parasite Cuscuta pentagona (dodder). The assembled transcriptome was used to analyze gene expression dynamic structure during dodder development and parasitism, and identified key gene categories involved in the process of plant parasitism. Host plant infection is accompanied with increased expression of parasite genes underlying transport and transporter categories, response to stress and stimuli, as well as genes encoding enzymes involved in cell wall modifications. By contrast expression of photosynthetic genes is decreased in the dodder infective stages compared to normal stem. In addition, genes relating to biosynthesis, transport and response of phytohormones, such as auxin, gibberellins and strigolactone, were differentially expressed in the dodder infective stages compared to stem and seedlings. Based on differential and coexpression gene analysis we prioritized LBD-like transcriptional regulator (LATERAL ORGAN BOUNDARY DOMAIN LIKE) from dodder transcripts for initial functional analysis. Specific fragment from the target gene was cloned into RNAi vectors under the 35S and the phloem specific SUC2 promoters and used to generate tomato host plants for cross-species silencing. Once T1 seeds were produced we evaluated the growth of C. pentagona on the RNAi inducing tomato host. This examination demonstrated reduced fitness and less haustorial development of the parasite on the transgenic plants and suggests that LBD25 may be a central regulator of haustoria development. To further analyze the parasite and host structure and specific expression changes between the two plants interface we used laser-capture microdissection (LCM) coupled with RNA-seq. Haustoria and host samples were collected from 3 developmental stages during the infection and RNA-seq was performed by Illumina sequencing. Reads were mapped to our de-novo assembled transcriptome and analyzed informatically using gene clustering, differential gene expression, coexpression network analysis and predictions of secreted proteins. This identified a set of genes specific to the infecting tissue and when taken together with the whole data, these were identified as playing a central role in the gene expression network regulating the parasitic process. A narrower list of 14 candidate genes for further functional analysis by host induced gene silencing in the parasitic weed. We chose to test 3 of the candidates by generating tomato RNAi lines and for rapid screening all genes using Arabidopsis thaliana as a host. When dodder was grown on the RNAi arabidopsis T2 plants we found that the parasite growth was impaired when a WRKY6-like transcription factor was targeted for silencing and improved when a small peptide CLV-like gene was targeted for silencing. Once validated this data can provide additional targets in the parasite that can be manipulated to facilitate crop resistance to the weed and shed light on the mode of action of this unique weed. To identify the mechanisms that are activated in tomato in response to attack of the parasitic weed we used the LCM collected RNA-seq data from the host side and complemented it with and additional time course RNA-seq of early attachment stages (days 1 to 4 after attachment). Sequence reads were mapped to the tomato transcriptome and analyzed for gene expression changes as we did for the parasite side. The bioinformatic analysis revealed the most expression changes in the host occurs at the early stage on attachment and seem to involve strong indication of the stress phytohormones salicylic acid and ethylene. We therefore tested if mutants in the signaling pathway of the later show less susceptibility to C. pentagona. We found it not to be the case which suggest that the ethylene pathway is not a major contributor to tomato resistance to dodder. Additionally, based on early gene expression changes in tomato after dodder attachment we identified identified candidate central regulators of the tomato defence response which include a WRKY transcription factor, a PR1-like pathogen response gene and 2 receptor type genes. To evaluate the involvement of these candidate genes in the tomato defence response we generated CRISPR/Cas9 construct to specifically target and mutate them in tomato. Transgenic plants are being produced and genotyped for mutation in the target loci. S. lycopersicum, N. benthamiana and S. habrochaites respond differently to infestation of C. pentagona. To shed light on natural resistant mechanism to parasitic plants we conducted RNA-seq analysis of dodder growing on the three different species. We collected host tissue next to attachment sites at the early stage of infection in which we identified most changes accord.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Sinha, NR. Gene network modules regulating natural diversity in leaf shape and in development, Invited Speaker, SolGenomics 2014, Bahia, Brazil, November 2014
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Sinha, NR. Characterizing the Parasitism Processin the Parasitic Plant Cuscuta, Invited Speaker, Weed Biology Symposium, 2015, Lexington, KY, January 2015.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Sinha, NR. Stresses and networks, Invited Speaker, BP Pal Chair�Lecture, IISC Bangalore, INDIA, September 2015.
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Progress 01/01/14 to 12/31/14
Outputs
Target Audience: Weed scientists, basic biologists interested in parasitic plants, applied scientists interested in crop productivity Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Three post-doctoral students and one graduate student were trained in molecular biology, genomics and bioinformatics during this reporting year. How have the results been disseminated to communities of interest? A publication providing a large amount of data to the interested communities was published. The investigator has made sure the publication is open access for the broadest possible dissemination, and reported the publication on Facebook and Twitter. What do you plan to do during the next reporting period to accomplish the goals? In the next reporting period we plan to include additional analysis of gene expression changes in the haustoria and a comprehensive list of candidate genes involved in both the parasitism and host response. Furthermore, we anticipate we will have initial analysis from silencing key genes in the parasite. We also plan to have RNA-seq analysis of dodder growing on S. lycopersicum, N. benthamiana and S. habrochaites which respond differently to infection which may shed light on potential natural resistant mechanism to parasitic plants.
Impacts
What was accomplished under these goals?
The molecular mechanisms underlying plant parasitism and host responses are poorly understood and likely involve plant-plant crosstalk and numerous genes/gene-regulatory changes. In order to investigate the process of parasitism, RNAs from different stages, i.e. seed, seedling, vegetative strand, prehaustoria, haustoria, and flowers, were used to de-novo assemble and annotate the transcriptome of the obligate plant stem parasite Cuscuta pentagona (dodder). The assembled transcriptome was used to analyze gene expression dynamic structure during dodder development and parasitism, and identified key gene categories involved in the process of plant parasitism. Host plant infection is accompanied with increased expression of parasite genes underlying transport and transporter categories, response to stress and stimuli, as well as genes encoding enzymes involved in cell wall modifications. By contrast expression of photosynthetic genes is decreased in the dodder infective stages compared to normal stem. In addition, genes relating to biosynthesis, transport and response of phytohormones, such as auxin, gibberellins and strigolactone, were differentially expressed in the dodder infective stages compared to stem and seedlings. To further analyze the parasite and host structure and specific expression changes and the two plants interface we used laser-capture microdissection (LCM) coupled with RNA-seq. Haustoria and host samples (4-7 biological replicates) were collected from 3 developmental stages during the infection and analyzed RNA-seq was preformed by sequencing 3 lanes of Illumina HiSeq 2000. Quality filtered reads were mapped to our de-novo assembled transcriptome and analyzed for gene expression. This identified a set of genes specific to the infecting tissue and when taken together with the whole data, these were identified as playing a central role in the gene expression network regulating the parasitic process. Differential and coexpression gene analysis provided a list of potential genes for further functional analysis by silencing in the parasitic weed. At this stage we prioritized two candidates from dodder transcripts including a (LATERAL ORGAN BOUNDARY DOMAIN LIKE) LBD-like transcriptional regulator and a sugar transporter-like. Specific fragments from the target genes were cloned into RNAi vectors under the 35S and/or the phloem specific SUC2 promoters and are being used to generate tomato host plants for cross-species silencing. Our investigation sheds light on the transcriptional changes that accompany plant parasitism and aids in identifying gene targets for use in controlling infestation of crops by parasitic weeds.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Ranjan, A., Ichihashi, Y., Farhi, M., Zumstein, K., Townsley, B., David-Schwartz, R and Sinha, NR. (2014) De novo assembly and characterization of the transcriptome of the parasitic weed Cuscuta pentagona identifies genes associated with plant parasitism. Plant Physiology. 166:1186-1199
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