GENOMIC AND GENETIC APPROACHES IN IDENTIFYING GENES THAT REGULATE STRAWBERRY FRUIT DEVELOPMENT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1010278
• Project No.: MD-CBMG-8881
• Project Start Date: Jul 20, 2016
• Project End Date: Aug 31, 2019

Project Director
Liu, ZH.

Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742

Performing Department
Cell Biology & Molecular Genetics

Non Technical Summary
Strawberry is an important specialty crop in US. The cultivated strawberry is octoploid (4 pairs of chromosomes for each of the 7 chromosomes) and is thus tremendously complex in its genome. We are utilizing Fragaria vesca, a wild and diploid strawberry, to study strawberry fruit development. This diploid strawberry is easy to grow and has already a sequenced genome. However, there has been no effective method to knockout genes in this wild strawberry, limiting its use as a model experimental system. We are pioneering the CRISPR/CAS9 genome editting method for strawberry and have had preliminarily success. We would like to improve this method further by adopting a virus-based delivery method. In addition, we would like to employ gene knockout methods to study a number of genes previously identified to be specifically expressed in the wild strawberry fleshy fruit (the receptacle). These analyses will shed light on the function of these fruit-specific genes. Combined, our efforts will provide much needed molecular insights into strawberry fruit development, based on which fruits with desirable traits and yields will be engeneered with the CRISPR/Cas9 genome editting tool.

Animal Health Component

0%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1122	1050	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1122 - Strawberry;

Field Of Science
1050 - Developmental biology;

Keywords

fruit development

diploid strawberry

crispr/cas9

Goals / Objectives
Goal#1: Use CRISPR, RNAi, and over-expression to determine the function of fruit-specific genes in strawberry.Goal#2: Further develop CRISPR as a gene editing tool in strawberry, including experimentations using a virus-mediated delivery method.

Project Methods
Research MethodsGoal1: Use CRISPR/Cas9, RNAi, and over-expression to determine the function of fruit-specific genes in strawberry. To determine the function of fruit-specific genes (ie. FT, auxin signaling genes and other genes of interests), we will both overexpress and knock down these genes of interest in F. vesca and examine the resulting plants for unusual fruit phenotypes. For overexpression, we will use the Gateway vector pMDC32 or pK7WG2D, which will allow constitutive expression under the control of the Cauliflower Mosaic virus 35S promoter(Curtis and Grossniklaus, 2003). To create gene knock downs, we will use both RNAi and CRISPR/Cas9. For RNAi, a gene-specific region of the exon will be cloned into the RNAi binary vector pK7GWIWG2 (I), the resulting construct will be introduced into strawberry plants through agrobacterium mediated transformation. The CRISPR/Cas9 is new method for targeted gene editing that relies on a short RNA sequence to guide the Cas9 endonuclease to a targeted site, which is subsequently cleaved and repaired through the error-prone Non-Homologous End Joining DNA repair mechanism. After repair, the targeted site will have lost or gained several bases, thereby rendering the gene non-functional (Doudna and Charpentier, 2014). The nature of any resulting overexpression or knock down phenotypes will allow us to gain knowledge both in the developmental process of fruit initiation and expansion and in understanding the regulatory roles of specific genes and pathways.Goal2: Further develop CRISPR/Cas9 as a gene-editing tool in strawberry, including experimentations using a virus-mediated delivery method.The type II CRISPR/Cas RNA-guided genome editing is the most recent addition to the tool kit of reverse genetics with additional advantages of low cost, easy to use, high efficiency. Further, this method makes it possible to derive transgene-free plants after the Cas9 construct is segregated away or when Cas9 is introduced via viruses. Although this technology has been demonstrated to work in Arabidopsis, Tabacco, and rice, it is not yet demonstrated in strawberry. The potential of this tool for targeted gene editing combined with the ability to obtain transgene-free plants in subsequent generations makes this technique extremely powerful and practical. We are very interested in adopting this technology for strawberry as it will empower us in basic research for gene function identification as well as allow us to more easily translate the research results into garden strawberry.At present, we have constructed several different Cas9/sgRNA cloning vectors, where the CAS9 nuclease gene is driven by 35S promoter, or ubiquitin promoter, or egg cell-specific promoter, or a strong promoter called YAO. In these vectors, the guide-RNA (gRNA) is driven by the Arabidopsis U6 promoter/terminator or by the strawberry (F. vesca) U6 promoter/terminator. We tested these vectors in two different ways. One is to test these vector in tobacco cells, where the sgRNA is designed to target a mutated GFP gene. If Cas9/sgRNA successfully mutated the GFP, a fraction of the GFP will be restored to wild type and will show green fluorescence. We have demonstrated the success of our vectors in restoring GFP fluorescence (Zhou and Liu, unpublished). The second method of testing is to knockdown the Phytoene Desaturase (PDS) gene in vivo in the wild strawverry. PDS gene is frequently used as a marker in Virus-Induced Gene Silensing (VIGS) (Velásquez et al., 2009). Knockdown of PDS results in white sectors on green tissues indicating successful reductions of PDS gene function. By Blast search of F. vesca genome, a single PDS gene has been identified. gRNA targeting the F. vesca PDS gene has been designed and preliminary success is shown by seeing white sectors in T1 generation transgenic strawberry plants (Zhou and Liu, unpublished).A Geminivirus system was previously shown to induce high efficiency genome editing (CRISPR-based) in Arabidopsis, Tobacco, and tomato without transgenesis (Cermak et al., 2015). We plan to adopt this system to deliver the CRISPR/CAS9 gene into the strawberry leaves and then generate calli from these leaves. If successful, we will be able to generate transgene-fee strawberries that can be more easily commercialized. Preliminary data expressing GFP within this virus-vector suggests that the this virus-vector can replicate within Fragaria vesca, indicating the feasibility of this approach in strawberry (Fischer and Liu, unpublished). To develop this tool in strawberry and to further Goal 1, the virus vector will be designed to carry the CRISPR/CAS9 cassette that cuts in the target gene (PDS as the first testing gene) to create knockouts. If successful, this method will be applied to knockout other genes described in goal1.

Progress 07/20/16 to 08/31/19

Outputs
Target Audience:Based on the CRISPR gene editing system we developed for strawberry, we successfully tested the function of several genes including ARF8 and GID1a (Zhou et al., 2020). We also wrote a review about CRISPR technology (Zhou et al., 2020).Finally, we identified a mutant in strawberry CUC2a gene with leaf and flower defects; in collaboration with Dr. Chunying Kang in Huazhong Agriculture University (China), we published this work together (Zheng et al., 2019).? Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A graduate student and a postdoc have contributed to the progress of the experiments and also learned how to write review articles and research manuscripts. How have the results been disseminated to communities of interest?The results have been published through peer-reviewed journals. Dr. ZhongchiLiu and her postdoc also spoke about their findings in scientific conferences such as the Plant and Animal Genome Conference in Jan. 2020 in San Diego, CA. 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 have accomplished Goal#1 and #2 by developing a CRISPR system for gene editing in strawberry and successfully applied this technology to test gene function in a number of fruit-expressed genes. However, we were unsuccessful in developing the virus-mediated delivery system. All of our studies relied on the agrobacterium-mediated gene delivery system.

Publications

Progress 10/01/18 to 08/31/19

Outputs
Target Audience:During this period, we have applied CRISPR/CAS9 technology to knockout several genes that may affect early strawberry fruit development. We have focused on genes in auxin signaling pathway including ARF6, ARF8 and IAA4. We have also attempted to knockout FT (Flower locus T), which show highly specific expression in strawberry receptacle fruit. At the moment, we have confirmed successful knockout of ARF8 and found that arf8 mutant fruit is slightly larger than the wild type. The work is being written up for a publication. For FT, we have obtained transgenic plants carrying the CAS9 constructs. We are in the process of identifying genome editing in the targeted loci of FT. Analysis of these CRISPR engineered plants will provide important molecular insights into how pollination and fertilization induce auxin hormone signaling and fruit enlargement. Two PhD students, Fuxi Wang and John Sittmann received training in working with strawberry genetics, including constructing CRISPR plasmid constructs, tissue culture, and transformation. Further, Fuxi Wang contributed to writing a review article on the application of CRISPR to fruit crops. She received training in scientific writing and literature reviews. Fuxi Wang also identified a mutant with leaf and flower defects. In collaboration with Dr. Chunying Kang in Huazhong Agriculture University (China), Fuxi Wang contributed to the identification of the causal mutation as a mutation in the CUC2 transcription factor gene. This work with Fuxi Wang as a co-author is now published. Graduate student John Sittmann received further training in characterising fruit phenotypes of mutant plants. John Sittmann recently defended his PhD thesis. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students were trained in strawberry genetics, gene cloning, transformation, and genome editing. One of the graduate students has successfully defended his PhD thesis. One undergraduate student Ms. Madison Plunkert learned the basic lab skills. How have the results been disseminated to communities of interest?The two graduate students have attended conferences and presented their work through posters. Manuscripts have been written and two are published. Dr. Liu has given seminars are several universities and one biotech company. 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 have shown successful genome editing (CRISPR) of strawberry genes in Fragaria vesca. We have also made several vectors suitable for CRISPR/Cas9 genome editing in strawberry. We have determined the function of ARF8 in fruit development using CRISPR and over-expression. We have in addition identified and characterized genes involved in strawberry leaf development using mutant screen and cloning.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Guanghui Zheng, Wei Wei, Yongping Li, Lijun Kan, Fuxi Wang, Xi Zhang, Feng Li, Zhongchi Liu, and Chunying Kang (2019) FvemiR164a acts both sequentially and redundantly with FveCUC2 in the formation of leaf serrations and floral organs in strawberry. New Phytologist Jun 10. doi: 10.1111/nph.15982. [Epub ahead of print]
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Junhui Zhou, Dongdong Li, Guoming Wang, Fuxi Wang, Merixia Kunjal, Dirk Joldersma, and Zhongchi Liu# (2019) Application and future perspective of CRISPR/Cas9 genome editing in fruit crops. Journal of Integrative Plant Biology Feb 21. doi: 10.1111/jipb.12793. [Epub ahead of print]
• Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Junhui Zhou, John Sittmann, Lei Guo, Xiaolong Huang, Anuhya Pulapaka and Zhongchi Liu (2020). Network-guided functional insights into auxin and GA crosstalk in strawberry fruit initiation.
• Type: Theses/Dissertations Status: Other Year Published: 2019 Citation: John Sittmann (2019) Investigating the regulation of growth mechanisms in two distinct branches of photosynthetic life. PhD dissertation.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:We have constructed several CRISPR/Cas9 vectors to knockout genes with potential roles in regulating fruit development (FT, ARFs, IAA, DELLA). Transforming these constructs into strawberry plants took a long time. We are still in the process of characterizing the transgenic plants and genotyping them to identify CRISPR-induced mutations. Graduate student Fuxi Wang was also involved in experiments to characterize Arabidopsis mutants defective in meristem development. Graduate student John Sittmann showed that a strawberry GA biosynthesis gene (GA20OX4) is able to rescue Arabidopsis ga20ox4 mutants. In contrast, a natural runnerless strawberry mutant has a 9bp deletion in the GA20OX gene, which was shown by John Sittmann to fail to rescue the Arabidopsis ga20ox4 mutant. The result indicates that the 9 bp deletion in the runnerless mutant of strawberry might be responsible for the runnerless phenotype. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students received training in strawberry and Arabidopsis transformation, plasmid construction, and other research techniques. How have the results been disseminated to communities of interest?Both graduate students have presented their work at local scientific conferences including Mid-Atlantic Section of SDB (Society of Developmental Biologist) conference and Mid-Atlantic Section of ASPB (American Society of Plant Biologists) conference. What do you plan to do during the next reporting period to accomplish the goals?We are hoping to characterize the transgenic plants, examine the effect of CRISPR-induced mutations on fruit development, and publish the work.

Impacts
What was accomplished under these goals? We are continuing our efforts to dissect strawberry gene function using CRISPR/Cas9, and RNAi.

Publications

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:In this time period, we have focused on identifying strawberry genes involved in ripening stage fruit development, especially transcription factors that regulate ripening. Transcription factors can regulate a network of genes involved in ripening; CRIPSR- or RNAi-based knockout or knockdown of a transcription factor will be more likely to cause a change in ripening characteristics. To start, we performed an RNA-sequencing experiment comparing green stage strawberry with turning stage (white stage) strawberry. This is a key stage when the fruit is transitioning into expressing ripening related genes. Analysis of the RNA-seq data allowed us to identify a MYB transcription factor named as MYB123a. In addition, we try to better visualize the RNA-sequancing data by creating an eFP browser hosted at Strawberry Genome Resources (SGR) website. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One visiting student received training in performing RNA extraction from fruits and RNA-seq. One PhD student, Charles Hawkins, received training and gained experiences in creating eFP browser. He also received his PhD degree partly based on this work. How have the results been disseminated to communities of interest?One paper is published. We also created a website where anyone could mine our RNA-seq data through eFP browser (http://mb3.towson.edu/efp/cgi-bin/efpWeb.cgi). Finally, we gave oral presentations at several conferences including Plant and Animal Genome Conference. What do you plan to do during the next reporting period to accomplish the goals?We plan to create transgenic plants that use CRISPR or RNAi to knockout MYB123a. We also plan to identify additional transcription factors that might interact with MYB123a.

Impacts
What was accomplished under these goals? In order to improve strawberry fruit charateristics, we need to first identify genes with key roles in regulating this process before using CRISPR, RNAi and over-expression to manipulate their function. To this end, we performed RNA-seq at the critical transition stage of fruit ripening. Analysis of the data identified the MYB123a transcription factor. To improve the utility of the RNA-seq data for the strawberry ripening field, we made our data available through creating an eFP browser, where anyone could search for strawberry gene expression during ripening stages.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Charles Hawkins, Julie Caruana, Jiaming Li, Omar Darwish, Chris Zawora, Jun Wu, Nadim Alkharouf, and Zhongchi Liu# (2017) An eFP Browser for Visualizing Strawberry Fruit and Flower Transcriptomes. Horticulture Research 4, Article�number:�17029 doi:10.1038/hortres.2017.29

Progress 07/20/16 to 09/30/16

Outputs
Target Audience:During the first year of the grant, we designed CRISPR/Cas9 constructs and tested virus-mediated transient expression of CRISPR constructs. We were successful in using CRISPR to correct a mutated MYB10 gene which caused yellow fruit color. We injected a CAS9, sgRNA, and wild type donor DNA for MYB10 (on a virus-based vector) into yellow fruit, and the resulting yellow fruit was able to develop red sectors indicating successful genome editing in some of the fruit cells. We were also successful to show trasient expression of a GFP protein in young strawberry leaves using the virus-based vector. However, the work has run into obstacles when we try to perform transient infection of seedlings and meristem cells. This later goal is necessary to induce heritable genomic changes. Changes/Problems:The virus-based system of introducing CRISPR/Cas9 components have run into problems (no succssful infection) when we tried to infect young seedlings and meristem cells, making it difficult to regenerate editted whole plants. What opportunities for training and professional development has the project provided?One graduate student, Matthew Fischer, received training in constructing CRISPR vectors and molecular cloning. One graduate student, Charles Hawkins, developed skills in bioinformatics. By comparing Fragaria vesca genome sequencing data, Charles identified a mutation in the FveMYB10 gene as the causal mutation for the yellow fruit color found in Yellow Wonder and Hawaii 4. How have the results been disseminated to communities of interest?Some of the results are published (see publication list). PI presented the fruit color work at conferences. 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 have made progress in applying CRISPR/Cas9 genome editting technology with a virus-based vector. The advantage of this virus-based vector is that the foreign DNA/genes are not integrated into the host genome and will be lost in subsequent cell divisions. Therefore, the resulting genome editted plants are transgene-free.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Charles Hawkins, Julie Caruana, Erin Schiksnis, and Zhongchi Liu# (2016) Genome-scale DNA variant analysis identifies a SNP that underlies the yellow fruit color in wild strawberry. Scientific Report Jul 5;6:29017. doi: 10.1038/srep29017.