Source: UNIVERSITY OF FLORIDA submitted to
CONSEQUENCES OF GENOME EDITING THE SUSCEPTIBILITY GENE CSLOB1 FOR CITRUS BACTERIAL CANKER
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1012394
Grant No.
2017-67013-26527
Project No.
FLA-CRC-005604
Proposal No.
2017-02986
Multistate No.
(N/A)
Program Code
A1171
Project Start Date
May 1, 2017
Project End Date
Apr 30, 2022
Grant Year
2017
Project Director
Wang, N.
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
IFAS
Non Technical Summary
With regard to genetic improvement for developing disease resistance, tree species in comparison to annuals represent a serious challenge. Citrus bacterial canker caused by Xanthomonas citri ssp. citri (Xcc) is one of the most devastating diseases of citrus in the world and developing resistance to this disease is a daunting task. Generating canker resistant citrus varieties would be the most sustainable and economical solution to control canker. However, citrus breeding has often been hindered by polyembryony, pollen-ovule sterility, sexual and graft incompatibilities, and extended juvenility. Here, we propose to exploit CRISPR technology and a specific disease-susceptibility gene in citrus to generate canker resistant plants. CsLOB1 (Citrus sinensis Lateral Organ Boundaries), which is a critical citrus susceptibility gene that the citrus canker pathogen Xcc targets, is a member of the Lateral Organ Boundaries Domain (LBD) gene family of transcription factors. All strains of Xcc encode a transcription activator-like (TAL) effector that recognizes an effector binding element (EBE) in the promoter of CsLOB1 and induces gene expression. Citrus canker represents a model disease for the assessment of genome editing for the enhancement of tree germplasm. The project will generate edited plants and assess the consequences of editing. Furthermore, experiments are proposed that will provide insight into the ability of the pathogen to overcome editing by adaptation. Accomplishments from this project will pave the way for generating non-transgenic plants in other tree crops for improving traits such as disease resistance, flowering time, and yield.
Animal Health Component
0%
Research Effort Categories
Basic
60%
Applied
30%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21209201100100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
0920 - Orange;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
We propose to exploit CRISPR technology and a specific disease-susceptibility gene in citrus to generate canker resistant plants. CsLOB1 (Citrus sinensis Lateral Organ Boundaries), which is a critical citrus susceptibility gene that the citrus canker pathogen Xanthomonas citri (Xcc) targets, is a member of the Lateral Organ Boundaries Domain (LBD) gene family of transcription factors. All strains of Xcc encode a transcription activator-like (TAL) effector that recognizes an effector binding element (EBE) in the promoter of CsLOB1 and induces gene expression. Citrus canker represents a model disease for the assessment of genome editing for the enhancement of tree germplasm. The project will generate edited plants and assess the consequences of editing. Furthermore, experiments are proposed that will provide insight into the ability of the pathogen to overcome editing by adaptation. Accomplishments from this project will pave the way for generating non-transgenic plants in other tree crops for improving traits such as disease resistance, flowering time, and yield.
Project Methods
Objective 1. Development of CBC resistant varieties by genome modification of the susceptibility gene CsLOB1 gene using CRISPR-Cas9 techniques. We will generate non-transgenic plant lines with EBE modifications in the promoter region of CsLOB1 in Valencia sweet orange through protoplast transformation using purified recombinant Cas9 protein and sgRNA and test the modified varieties against a worldwide Xcc strain collection.Objective 2. Measuring the effects of the mutations on CsLOB1 expression, citrus development, and other horticultural traits. We will analyze the expression properties of CsLOB1 in normal, null, and edited plants using quantitative real time reverse transcription PCR (qRT-PCR) of tissues and CsLOB1 promoter regions fused to the beta-glucuronidase (GUS) gene. We will measure physiological parameters of plant development and other horticultural traits of the null, promoter-edited and normal plants.Objective 3. Assessing the ability of Xcc to adapt to resistant plants and TAL effector-deficient strains to adapt to susceptible plants. We will determine the ability of Xcc strains that are defective for CsLOB1 induction to re-acquire pustule formation on normal plants (using TAL effector gene mutant strains) and on edited plant lines (using wild type strains). We will determine the potential for additional LBD genes to support pustule formation in citrus bacterial canker using dTALes that target selected LBD promoters.

Progress 05/01/17 to 04/30/22

Outputs
(N/A)

Impacts
What was accomplished under these goals? Aim 1. Development of CBC resistant varieties by genome modification of the susceptibility gene CsLOB1 gene using CRISPR-Cas9 techniques. In previous studies, canker-resistant plants were generated by mutating the coding region or the EBE ofLOB1. However, homozygous or biallelic canker-resistant plants have not been generated for commercial citrus varieties, such as grapefruit (Citrus paradisi), which usually contain two alleles ofLOB1and thus, have two types ofLOB1promoter sequences: TI LOBP and TII LOBP. Two different sgRNAs were used to target both EBE types. Both 35S promoter and Yao promoter were used to drive the expression of SpCas9p to modify EBEPthA4-LOBP in grapefruit. Using 'Duncan' grapefruit epicotyls as explants, 19 genome-edited grapefruit plants were generated with one biallelic mutant line (#DunYao7).X. citrisubsp.citricaused canker symptoms on wild-type and nonbiallelic mutant plants but not on #DunYao7. XccPthA4 mutant containing the designer TAL effector dLOB1.5, which recognizes a conserved sequence in both wild-type and #DunYao7, caused canker symptoms on both wild-type and #DunYao7. No off-target mutations were detected in #DunYao7. This study represents the first time that CRISPR-mediated genome editing has been successfully used to generate disease-resistant plants for 'Duncan' grapefruit, paving the way for using disease-resistant varieties to control canker. Here, we have successfully adapted the ABE to edit the TATA box in the promoter region of the canker susceptibility geneLOB1from TATA to CACA in grapefruit (Citrus paradise) and sweet orange (Citrus sinensis). TATA-edited plants are resistant to the canker pathogenXanthomonas citrisubsp.citri(Xcc). Editing the EBE region with Cas9/gRNA has been used to generate canker resistant citrus plants. However, most of the EBE-edited lines generated contain indels of 1-2 bp, which has higher possibility to be overcome by PthA4 adaptation. The adaptation capacity of TALEs inversely correlates with the number of mismatches with the EBE. LbCas12a/crRNA is known to generate longer deletion than Cas9. In this study, we used a temperature-tolerant and more efficient LbCas12a variant (ttLbCas12a), harboring the single substitution D156R, to modify the EBE region ofLOB1. We first constructed GFP-p1380N-ttLbCas12a:LOBP, which was shown to be functional via Xcc-facilitated agroinfiltration in Pummelo (Citrus maxima) leaves. Subsequently, we stably expressed ttLbCas12a:LOBP in Pummelo. Eight transgenic lines were generated, with seven lines showing 100% mutations of the EBE, among which one line is homozygous. The EBE-edited lines had the ttLbCas12a-mediated deletions of up to 10 bp. Importantly, the seven lines were canker resistant and no off-targets were detected. In summary, ttLbCas12a can be used to efficiently generate biallelic/homozygous citrus mutant lines with short deletions, thus providing a useful tool for the functional study and breeding of citrus. Generation of biallelic/homozygous mutants remains difficult for sweet orange due to low transformation rate, existence of heterozygous alleles for target genes, and low biallelic editing efficacy using the CRISPR technology. Here, we report improvements in the CRISPR/Cas9 system for citrus gene editing. Based on the improvements we made previously [dicot codon optimized Cas9, tRNA for multiplexing, a modified sgRNA scaffold with high efficiency, citrus U6 (CsU6) to drive sgRNA expression], we further improved our CRISPR/Cas9 system by choosing superior promoters [Cestrum yellow leaf curling virus(CmYLCV) orCitrus sinensisubiquitin (CsUbi) promoter] to drive Cas9 and optimizing culture temperature. This system was able to generate a biallelic mutation rate of up to 89% for Carrizo citrange and 79% for Hamlin sweet orange. Consequently, this system was used to generate canker-resistant Hamlin sweet orange by mutating the effector binding element (EBE) of canker susceptibility geneCsLOB1, which is required for causing canker symptoms byXcc. Six biallelic Hamlin sweet orange mutant lines in the EBE were generated. The biallelic mutants are resistant toXcc. Biallelic mutation of the EBE region abolishes the induction ofCsLOB1byXcc. This study represents a significant improvement in sweet orange gene editing efficacy and generating disease-resistant varietiesviaCRISPR-mediated genome editing. This improvement in citrus genome editing makes genetic studies and manipulations of sweet orange more feasible. Aim 2. Measuring the effects of the mutations on CsLOB1 expression, citrus development, and other horticultural traits. The homozygous plants have been evaluated for the effects of the mutations on CsLOB1 expression, citrus development, and other horticultural traits. No side effects were observed for the three homozygous or biallelic Citrus maxima or C. citri. No off-targets were identified. Aim 3. Assessing the ability of Xcc to adapt to resistant plants and TAL effector-deficient strains to adapt to susceptible plants. We investigated the consequences of TALE adaptations in the citrus pathogen Xanthomonas citri subsp. citri (Xcc), in which PthA4 is the TALE required for pathogenicity, whereas CsLOB1 is the corresponding susceptibility gene, on host resistance. Seven TALEs, containing two-to-nine mismatching-repeats to the EBEPthA4 that were unable to induce CsLOB1 expression, were introduced into Xcc pthA4:Tn5 and adaptation was simulated by repeated inoculations into and isolations from sweet orange for a duration of 30 cycles. While initially all strains failed to promote disease, symptoms started to appear between 9- 28 passages in four TALEs, which originally harbored two-to-five mismatches. Sequence analysis of adapted TALEs identified deletions and mutations within the TALE repeat regions which enhanced putative affinity to the CsLOB1 promoter. Sequence analyses suggest that TALEs adaptations result from recombinations between repeats of the TALEs. Reintroduction of these adapted TALEs into Xcc pthA4:Tn5 restored the ability to induce the expression of CsLOB1, promote disease symptoms and colonize host plants. TALEs harboring seven-to-nine mismatches were unable to adapt to overcome the incompatible interaction. Our study experimentally documented TALE adaptations to incompatible EBE and provided strategic guidance for generation of disease resistant crops against TALE-dependent pathogens. We have inoculated the homozygous canker resistant citrus varieties pummelo with Xcc and investigated whether Xcc is able to adapt to the mutants.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Jia H, Omar AA, Orbovi? V, Wang N. Biallelic Editing of the LOB1 Promoter via CRISPR/Cas9 Creates Canker-Resistant 'Duncan' Grapefruit. Phytopathology. 2022 Feb;112(2):308-314.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Teper D, Xu J, Pandey SS, Wang N. PthAW1, a Transcription Activator-Like Effector of Xanthomonas citri subsp. citri, Promotes Host-Specific Immune Responses. Mol Plant Microbe Interact. 2021 Sep;34(9):1033-1047.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Huang X, Wang Y, Wang N. Base Editors for Citrus Gene Editing. Front Genome Ed. 2022 Feb 28;4:852867. doi: 10.3389/fgeed.2022.852867.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Teper D, Pandey SS, Wang N. The HrpG/HrpX Regulon of Xanthomonads-An Insight to the Complexity of Regulation of Virulence Traits in Phytopathogenic Bacteria. Microorganisms. 2021 Jan 16;9(1):187.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Jia H, Wang Y, Su H, Huang X, Wang N. LbCas12a-D156R Efficiently Edits LOB1 Effector Binding Elements to Generate Canker-Resistant Citrus Plants. Cells. 2022 Jan 18;11(3):315.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Huang X, Wang Y, Wang N. Highly Efficient Generation of Canker-Resistant Sweet Orange Enabled by an Improved CRISPR/Cas9 System. Front Plant Sci. 2022 Jan 11;12:769907.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Zhang Y, Andrade MO, Wang W, Teper D, Romeo T, Wang N. Examination of the Global Regulon of CsrA in Xanthomonas citri subsp. citri Using Quantitative Proteomics and Other Approaches. Mol Plant Microbe Interact. 2021 Nov;34(11):1236-1249.


Progress 05/01/20 to 04/30/21

Outputs
Target Audience:Scientific community, citrus growers, graduate students, agricultural industry Changes/Problems:COVID19 slowed down our progress, but we are able to complete the project as planned. What opportunities for training and professional development has the project provided?Training was provided for 4 undergrads and 5 graduate students (including 2 visiting students from Brazil), and 2 postdoctoral fellows. How have the results been disseminated to communities of interest?The results have been published in6manuscripts,two protocols, one industry articles, 3 abstractspresented in meetings andtwoinvited seminars. What do you plan to do during the next reporting period to accomplish the goals?Continue optimizing non-transgenic genome editing technology Continue generating non-transgenic canker resistant citrus varieties. Evaluate the effects of the mutations on CsLOB1 expression, citrus development, and other horticultural traits. Assessing the ability of Xcc to adapt to resistant plants and TAL effector-deficient strains to adapt to susceptible plants.

Impacts
What was accomplished under these goals? Aim 1. Development of CBC resistant varieties by genome modification of the susceptibility gene CsLOB1 gene using CRISPR-Cas9 techniques. Herer, we employed CRISPR-LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND2006, to edit a citrus genome for the first time. First, LbCas12a was used to modify the CsPDS gene successfully in Duncan grapefruit via Xcc- facilitated agroinfiltration. Next, LbCas12a driven by either the 35S or Yao promoter was used to edit the PthA4 effector binding elements in the promoter (EBEP thA4 -CsLOBP) of CsLOB1. A single crRNA was selected to target a conserved region of both Type I and Type II CsLOBPs, since the protospacer adjacent motif of LbCas12a (TTTV) allows crRNA to act on the conserved region of these two types of CsLOBP. CsLOB1 is the canker susceptibility gene, and it is induced by the corresponding pathogenicity factor PthA4 in Xanthomonas citri by binding to EBEP thA4 -CsLOBP. A total of seven 35S- LbCas12a-transformed Duncan plants were generated, and they were designated as #D35 s1 to #D35 s7, and ten Yao- LbCas12a-transformed Duncan plants were created and designated as #Dyao 1 to #Dyao 10. LbCas12a-directed EBEP thA4 - CsLOBP modifications were observed in three 35S-LbCas12a-transformed Duncan plants (#D35 s1, #D35 s4 and #D35 s7). However, no LbCas12a-mediated indels were observed in the Yao-LbCas12a-transformed plants. Notably, transgenic line #D35 s4, which contains the highest mutation rate, alleviates XccΔpthA4:dCsLOB1.4 infection. Finally, no potential off-targets were observed. Plant codon-optimized CRISPR-SpCas9 was employed to disrupt the PthA4 effector binding elements in the promoter (EBEPthA4-LOBP) of the canker susceptibility gene LOB1 of Pummelo (Citrus maxima). Citrus canker is caused by Xanthomonas citri subsp. citri (Xcc). Citrus canker disease symptoms are elicited through inducing LOB1 by PthA4, a type III secretion effector in the transcriptional activator like effector family, recognizing EBEPthA4-LOBP. A single sgRNA was designed to modify two alleles of EBEPthA4-LOBP in Pummelo, a pure citrus species containing two identical alleles of EBEPthA4-LOBP. Xcc-facilitated agroinfiltration was initially employed to test and confirmed the function of the binary vector GFP-p1380N-SpCas9p:PumLOBP constructed to target the EBEPthA4-LOBP. The binary vector was used to transform Pummelo. Totally, eight transgenic lines were generated, designated as #Pum1 to #Pum8. All of transgenic lines had targeted modifications in the EBEPthA4-LOBP. Among them, #Pum1 is a homozygous mutant and #Pum6 is a biallelic mutant. Both #Pum1 and #Pum6 fully resist against Xcc infection. No off-targets were found in #Pum1. We have recently generated one homozygous mutant for Hamlin sweet orange with an adenine insertion within EBEPthA4?LOBP. We have developed multiplex genome editing toolkits for citrus that significantly improve citrus genome editing efficacy. CRISPR/Cas systems have been engineered for genome editing in many organisms, including plants. However, the gene editing efficiency in citrus via CRISPR technology remains too low to be implemented for genetic improvement in practice. Moreover, it is very difficult to obtain homozygous or biallelic knockout mutants in citrus. Here, we have developed multiplex genome editing toolkits for citrus including PEG-mediated protoplast transformation, a GFP reporter system that allows the rapid assessment of CRISPR constructs, citrus U6 promoters with improved efficacy, and tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 44.4% and a homozygous mutation rate of 11.1%, representing a significant improvement in citrus genome editing efficacy. In addition, our study lays the foundation for nontransgenic genome editing of citrus. Meiwa kumquat (Fortunella crassifolia) has shown a durable resistance against Xcc. Here, we aimed to characterize the mechanisms responsible for such a durable resistance by characterizing the transcriptional and physiological responses of Meiwa kumquat to Xcc. Inoculation of Meiwa kumquat with Xcc promoted immune responses such as upregulation of PR genes, accumulation of salicylic acid, hypersensitive response (HR)-like cell death and early leaf abscission. Hypertrophy and hyperplasia symptoms, which are known to be caused by Xcc-induction of the canker susceptibility gene LOB1 through the transcription activator-like effector (TALE) PthA4, always appear prior to the development of cell death. Mutation of pthA4 in Xcc abolished the induction of LOB1, canker symptoms, cell death, and leaf abscission and reduced the expression of PR genes in inoculated kumquat leaves without reducing Xcc titers in planta. Transcriptome analysis demonstrated that PthA4 promotes plant biotic and abiotic stress responses and the biosynthesis of abscisic acid. Transcriptional induction of LOB1 homologs in Meiwa kumquat by Xcc pthA4 mutant strains carrying a repertoire of designer TALEs promoted the elicitation of HR-like phenotype and leaf abscission, suggesting that kumquat response to Xcc is associated with upregulation of LOB1. Our study suggests a novel mechanism of plant resistance to Xanthomonas via elicitation of immune responses by upregulation of a host susceptibility gene. Aim 2. Measuring the effects of the mutations on CsLOB1 expression, citrus development, and other horticultural traits. The homozygous plants are being evaluated for the effects of the mutations on CsLOB1 expression, citrus development, and other horticultural traits. For the mutants that are not homozygous, no obvious side effects were observed. No side effects were observed for the three homozygous or biallelic Citrus maxima or C. citri. No off-targets were identified. Aim 3. Assessing the ability of Xcc to adapt to resistant plants and TAL effector-deficient strains to adapt to susceptible plants. We investigated the consequences of TALE adaptations in the citrus pathogen Xanthomonas citri subsp. citri (Xcc), in which PthA4 is the TALE required for pathogenicity, whereas CsLOB1 is the corresponding susceptibility gene, on host resistance. Seven TALEs, containing two-to-nine mismatching-repeats to the EBEPthA4 that were unable to induce CsLOB1 expression, were introduced into Xcc pthA4:Tn5 and adaptation was simulated by repeated inoculations into and isolations from sweet orange for a duration of 30 cycles. While initially all strains failed to promote disease, symptoms started to appear between 9-28 passages in four TALEs, which originally harbored two-to-five mismatches. Sequence analysis of adapted TALEs identified deletions and mutations within the TALE repeat regions which enhanced putative affinity to the CsLOB1 promoter. Sequence analyses suggest that TALEs adaptations result from recombinations between repeats of the TALEs. Reintroduction of these adapted TALEs into Xcc pthA4:Tn5 restored the ability to induce the expression of CsLOB1, promote disease symptoms and colonize host plants. TALEs harboring seven-to-nine mismatches were unable to adapt to overcome the incompatible interaction. Our study experimentally documented TALE adaptations to incompatible EBE and provided strategic guidance for generation of disease resistant crops against TALE-dependent pathogens. We are inoculating the homozygous canker resistant pummelo with Xcc to see whether Xcc will be able to adapt. This experiment is ongoing.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: An, S.Q., Potnis, N., Dow, M., Vorh�lter, F.J., He, Y.Q., Becker, A., Teper, D., Li, Y., Wang, N., Bleris, L., and Tang, J.L. 2020. Mechanistic insights into host adaptation, virulence and epidemiology of the phytopathogen Xanthomonas. FEMS Microbiol Rev 44:1-32.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Teper, D., Xu, J., Li, J., and Wang, N. 2020. The immunity of Meiwa kumquat against Xanthomonas citri is associated with a known susceptibility gene induced by a transcription activator-like effector. PLoS Pathog 16:e1008886
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Pena, M.M., Teper, D., Ferreira, H., Wang, N., Sato, K.U., Ferro, M.I.T., and Ferro, J.A. 2020. mCherry fusions enable the subcellular localization of periplasmic and cytoplasmic proteins in Xanthomonas sp. PLoS One 15:e0236185.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Jia, H., and Wang, N. 2020. Generation of homozygous canker-resistant citrus in the T0 generation using CRISPR-SpCas9p. Plant Biotechnol J. 18:1990-2
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Huang, X., Wang, Y., Xu, J., and Wang, N. 2020. Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations. Plant Mol Biol 104:297-307
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Doron Teper Nian Wang 2021 Consequences of adaptation of TAL effectors on host susceptibility to Xanthomonas PLOS Genetics 17(1):e1009310


Progress 05/01/19 to 04/30/20

Outputs
Target Audience:Scientific community, citrus growers, graduate students Changes/Problems:COVID19 has significantly affected our research including slowing down the progress, and travel plan to present the research progress. What opportunities for training and professional development has the project provided?We trained one Ph.D. student and two postdocs in genome editing and canker research. How have the results been disseminated to communities of interest?The results have been published in 5 manuscripts, presented in meetings and invited seminars. What do you plan to do during the next reporting period to accomplish the goals?1. Continue to generating non-transgenic canker resistant citrus varieties. 2. Evaluate the effects of the mutations on CsLOB1 expression, citrus development, and other horticultural traits. 3. Assessing the ability of Xcc to adapt to resistant plants and TAL effector-deficient strains to adapt to susceptible plants.

Impacts
What was accomplished under these goals? Aim 1. Development of CBC resistant varieties by genome modification of the susceptibility gene CsLOB1 gene using CRISPR-Cas9 techniques. Herer, we employed CRISPR-LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND2006, to edit a citrus genome for the first time. First, LbCas12a was used to modify the CsPDS gene successfully in Duncan grapefruit via Xcc-facilitated agroinfiltration. Next, LbCas12a driven by either the 35S or Yao promoter was used to edit the PthA4 effector binding elements in the promoter (EBEPthA4-CsLOBP) of CsLOB1. A single crRNA was selected to target a conserved region of both Type I and Type II CsLOBPs, since the protospacer adjacent motif of LbCas12a (TTTV) allows crRNA to act on the conserved region of these two types of CsLOBP. CsLOB1 is the canker susceptibility gene, and it is induced by the corresponding pathogenicity factor PthA4 inXanthomonascitri by binding to EBEPthA4-CsLOBP. A total of seven 35S-LbCas12a-transformed Duncan plants were generated, and they were designated as #D35s1 to #D35s7, and ten Yao-LbCas12a-transformed Duncan plants were created and designated as #Dyao1 to #Dyao10. LbCas12a-directed EBEPthA4-CsLOBP modifications were observed in three 35S-LbCas12a-transformed Duncan plants (#D35s1, #D35s4 and #D35s7). However, no LbCas12a-mediated indels were observed in the Yao-LbCas12a-transformed plants. Notably, transgenic line #D35s4, which contains the highest mutation rate, alleviates XccΔpthA4:dCsLOB1.4 infection. Finally, no potential off-targets were observed. Plant codon-optimized CRISPR-SpCas9 was employed to disrupt the PthA4 effector binding elements in the promoter (EBEPthA4-LOBP) of the canker susceptibility gene LOB1 of Pummelo (Citrus maxima). Citrus canker is caused by Xanthomonas citri subsp. citri (Xcc). Citrus canker disease symptoms are elicited through inducing LOB1 by PthA4, a type III secretion effector in the transcriptional activator like effector family, recognizing EBEPthA4-LOBP. A single sgRNA was designed to modify two alleles of EBEPthA4-LOBP in Pummelo, a pure citrus species containing two identical alleles of EBEPthA4-LOBP. Xcc-facilitated agroinfiltration was initially employed to test and confirmed the function of the binary vector GFP-p1380N-SpCas9p:PumLOBP constructed to target the EBEPthA4-LOBP. The binary vector was used to transform Pummelo. Totally, eight transgenic lines were generated, designated as #Pum1 to #Pum8. All of transgenic lines had targeted modifications in the EBEPthA4-LOBP. Among them, #Pum1 is a homozygous mutant and #Pum6 is a biallelic mutant. Both #Pum1 and #Pum6 fully resist against Xcc infection. No off-targets were found in #Pum1. ?Aim 2. Measuring the effects of the mutations on CsLOB1 expression, citrus development, and other horticultural traits. The homozygous plants are being evaluated for the effects of the mutations on CsLOB1 expression, citrus development, and other horticultural traits. For the mutants that are not homozygous, no obvious side effects were observed. Aim 3. Assessing the ability of Xcc to adapt to resistant plants and TAL effector-deficient strains to adapt to susceptible plants. We utilized an experimental evolution approach to mimic host adaptation of Xcc pthA4 mutant to sweet orange. We manufactured three "non-adapted" TALEs containing mismatching repeats to the EBEPthA4 that were unable to induce the transcription of CsLOB1. The TALEs were introduced into Xcc pthA4:Tn5 to repeatedly infect and be re-isolated from sweet orange. This experiment is ongoing. We are inoculating the homozygous canker resistant pummelo with Xcc to see whether Xcc will be able to adapt. This experiment is ongoing.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Diffusible signal factor (DSF)-mediated quorum sensing modulates expression of diverse traits in Xanthomonas citri and responses of citrus plants to promote disease. Li L, Li J, Zhang Y, Wang N. BMC Genomics. 2019 Jan 17;20(1):55. doi: 10.1186/s12864-018-5384-4.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: CRISPR-LbCas12a-mediated modification of citrus. Jia H, Orbovi? V, Wang N. Plant Biotechnol J. 2019 Oct;17(10):1928-1937.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: TfmR, a novel TetR-family transcriptional regulator, modulates the virulence of Xanthomonas citri in response to fatty acids. Teper D, Zhang Y, Wang N. Mol Plant Pathol. 2019 May;20(5):701-715.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Origin and diversification of Xanthomonas citri subsp. citri pathotypes revealed by inclusive phylogenomic, dating, and biogeographic analyses. Patan� JSL, Martins J Jr, Rangel LT, Belasque J, Digiampietri LA, Facincani AP, Ferreira RM, Jaciani FJ, Zhang Y, Varani AM, Almeida NF, Wang N, Ferro JA, Moreira LM, Setubal JC. BMC Genomics. 2019 Sep 9;20(1):700. doi: 10.1186/s12864-019-6007-4.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Stringent response regulators (p)ppGpp and DksA positively regulate virulence and host adaptation of Xanthomonas citri. Zhang Y, Teper D, Xu J, Wang N. Mol Plant Pathol. 2019 Nov;20(11):1550-1565.


Progress 05/01/18 to 04/30/19

Outputs
Target Audience:citrus growers, researchers, students, and consumers Changes/Problems:No What opportunities for training and professional development has the project provided?We trained one Ph.D. student and two postdocs. How have the results been disseminated to communities of interest?publications, presentation, and patent application. What do you plan to do during the next reporting period to accomplish the goals?We will contine to conduct the research as outlined in the timeline.

Impacts
What was accomplished under these goals? Xanthomonas citrissp. citri(Xcc) is an important plant?pathogenic bacterium that causes citrus canker disease worldwide. PthA, a transcriptional activator?like (TAL) effector, directs the expression of the canker susceptibility geneCsLOB1. Here, we report our recent progress in the functional characterization of CsLOB1. Subcellular localization analysis of CsLOB1 protein in citrus protoplast revealed that CsLOB1 is primarily localized in the nucleus. We showed thatCsLOB1expression driven by dexamethasone (DEX) inCsLOB1?GRtransgenic plants is associated with pustule formation following treatment with DEX. Pustule formation was not observed in DEX?treated wild?type plants and in non?treatedCsLOB1?GRtransgenic plants. Water soaking is typically associated with symptoms of citrus canker. Weaker water soaking was observed with pustule formation inCsLOB1?GRtransgenic plants following DEX treatment. WhenCsLOB1?GR?transgenic Duncan grapefruit leaves were inoculated with Xcc306ΔpthA4 and treated with DEX, typical canker symptoms, including hypertrophy, hyperplasia and water soaking symptoms, were observed on DEX?treated transgenic plant leaves, but not on mock?treated plants. Twelve citrus genes that are induced by PthA4 are also stimulated by the DEX?induced expression of CsLOB1. As CsLOB1 acts as a transcriptional factor, we identified putative targets of CsLOB1 via bioinformatic and electrophoretic mobility shift assays. Cs2g20600, which encodes a zinc finger C3HC4?type RING finger protein, has been identified to be a direct target of CsLOB1. This study advances our understanding of the function of CsLOB1 and the molecular mechanism of how Xcc causes canker symptoms via CsLOB1. Here, we conducted nontransgenic genome editing of citrus via the transient introduction of Cas9-sgRNA DNA, RNA or ribonucleoprotein (RNP) into protoplast by PEG-mediated transfection or into embryogenic cells in a suspension culture by particle bombardment. Citrus protoplast or embryogenic cells appear to be recalcitrant to transformation using different approaches. Only the transient expression of CRISPR/Cas9 DNA in protoplast cells led to efficient transformation, and a genome editing mutation rate of 5.7% was observed. We further used hiTAIL-PCR analysis to show that the genome-modified citrus cells are free of foreign DNA. We also detected low off-target mutations in genome-modified citrus cells. Our findings represent a breakthrough in citrus breeding using CRISPR technology. In addition, we employed CRISPR-LbCpf1, derived from Lachnospiraceae bacterium ND2006, to edit the citrus genome. First, LbCpf1 was successfully used to modify the CsPDS gene in grapefruit Duncan via the Xcc-facilitated agroinfiltration. Next, LbCpf1 driven by either 35S or Yao promoter was used to edit the PthA4 effector binding elements in the promoter (EBEPthA4-CsLOBP) of CsLOB1. CsLOB1 is the canker susceptibility gene induced by the corresponding pathogenicity factor PthA4 of Xanthomonas citri, via binding to EBEPthA4-CsLOBP. Totally, seven 35S-LbCpf1-transformed Duncan plants were generated, designated as #D35s1 to #D35s7, and ten Yao-LbCpf1-transformed Duncan plants were created, designated as #DYao1 to #DYao10. LbCpf1-directed EBEPthA4-CsLOBP modifications were observed in three 35S-LbCpf1-transformed Duncan (#D35s1, #D35s4 and #D35s7). However, no LbCpf1-mediated indels were observed in the Yao-LbCpf1-transformed plants. Importantly, transgenic line #D35s4, containing the highest mutation rate, alleviates XccΔpthA4:dCsLOB1.4 infection. Therefore, CRISPR-LbCpf1 can be readily used as a powerful tool for citrus genome editing. Most pathogenic bacteria deliver virulence factors into host cytosol through type III secretion systems (T3SS) to perturb host immune responses. The expression of T3SS is often repressed in rich medium but is specifically induced in the host environment. The molecular mechanisms underlying host-specific induction of T3SS expression is not completely understood. Here we demonstrate inXanthomonas citrithat host-induced phosphorylation of the ATP-dependent protease Lon stabilizes HrpG, the master regulator of T3SS, conferring bacterial virulence. Ser/Thr/Tyr phosphoproteome analysis revealed that phosphorylation of Lon at serine 654 occurs in the citrus host. In rich medium, Lon represses T3SS by degradation of HrpG via recognition of its N terminus. Genetic and biochemical data indicate that phosphorylation at serine 654 deactivates Lon proteolytic activity and attenuates HrpG proteolysis. Substitution of alanine for Lon serine 654 resulted in repression of T3SS gene expression in the citrus host through robust degradation of HrpG and reduced bacterial virulence. Our work reveals a novel mechanism for distinct regulation of bacterial T3SS in different environments. Additionally, our data provide new insight into the role of protein posttranslational modification in the regulation of bacterial virulence.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhou, X., Teper, D., Andrade, M.O., Zhang, T., Chen, S., Song, W.Y., and Wang, N. 2018. A Phosphorylation Switch on Lon Protease Regulates Bacterial Type III Secretion System in Host. MBio 9. Duan, S., Jia, H., Pang, Z., Teper, D., White, F., Jones, J., Zhou, C., and Wang, N. 2018. Functional characterization of the citrus canker susceptibility gene CsLOB1. Mol Plant Pathol. doi: 10.1111/mpp.12667. [Epub ahead of print] Ference, C.M., Gochez, A.M., Behlau, F., Wang, N., Graham, J.H., and Jones, J.B. 2018. Recent advances in the understanding of Xanthomonas citri ssp. citri pathogenesis and citrus canker disease management. Mol Plant Pathol 19:1302-1318. Gochez, A.M., Huguet-Tapia, J.C., Minsavage, G.V., Shantaraj, D., Jalan, N., Strau�, A., Lahaye, T., Wang, N., Canteros, B.I., Jones, J.B., and Potnis, N. 2018. Pacbio sequencing of copper-tolerant Xanthomonas citri reveals presence of a chimeric plasmid structure and provides insights into reassortment and shuffling of transcription activator-like effectors among X. citri strains. BMC Genomics 19:16. Riera, N., Wang, H., Li, Y., Li, J., Pelz-Stelinski, K., and Wang, N. 2018. Induced systemic resistance against citrus canker disease by rhizospheric bacteria. Phytopathology. doi.org/10.1094/PHYTO-07-17-0244-R
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Doron Teper, Yanan Zhang, Nian Wang. 2019. TfmR, a novel TetR?family transcriptional regulator, modulates the virulence of Xanthomonas citri in response to fatty acids. Mol Plant Pathol. doi: 10.1111/mpp.12786.


Progress 05/01/17 to 04/30/18

Outputs
Target Audience:Scientific community, graduate students, citrus growers, and goverment agencies Changes/Problems:There is no major problems. What opportunities for training and professional development has the project provided?This project has trained two Ph.D. students and two postdocs in studies related to citrus canker and genome editing. How have the results been disseminated to communities of interest?Yes. Three manuscripts have been submitted and published. Two posters have been represented in scientific meetings. In addition, one more manuscript is being prepared. What do you plan to do during the next reporting period to accomplish the goals?1. Continue to generate CsLOB1 mutated citrus using the CRISPR technology 2. Continue to testing the evolution of different X. citri strains in citrus showing compatible and incompatible interactions. 3. Functionally characterize CsLOB1 and its homologs. 4. Continue to characterize PthA and other virulence factors of Xanthomonas citri

Impacts
What was accomplished under these goals? To edit citrus genome by using CRISPR To edit the EBE region of CsLOB1, we have been using SpCas9, which only has one suitable target site in this region. To expand the target selection, we edit citrus genome using different Cas proteins. SaCas9/sgRNA, derived fromStaphylococcus aureus, is an alternative system for genome editing toStreptococcus pyogenesSpCas9/sgRNA. The smaller SaCas9 recognizes a different protospacer adjacent motif (PAM) sequence from SpCas9. SaCas9/sgRNA has been employed to edit the genomes ofArabidopsis, tobacco and rice. In this study, we aimed to test its potential in genome editing of citrus. Transient expression of SaCas9/sgRNA in Duncan grapefruit via Xcc-facilitated agroinfiltration showed it can successfully modifyCsPDSandCs2g12470. Subsequently, binary vector GFP-p1380N-SaCas9/35S-sgRNA1:AtU6-sgRNA2 was developed to edit two target sites ofCs7g03360in transgenic Carrizo citrange. Twelve GFP-positive Carrizo transformants were successfully established, designated as #Cz1 to #Cz12. Based on targeted next generation sequencing results, the mutation rates for the two targets ranged from 15.55 to 39.13% for sgRNA1 and 49.01 to 79.67% for sgRNA2. Therefore, SaCas9/sgRNA can be used as an alternative tool to SpCas9/sgRNA for citrus genome editing. In addition, we also tested Cpf1. Currently, non-transgenic genome editing approaches are being used to generate CsLOB1 modified citrus varieties. Test how X. citri evolves and whether they can overcome plant defense TAL effectors show very high similarity to each other in DNA and protein sequences. However, small changes in the variable regions in each repeat dictated the target specificity of each TAL variant. Our hypothesis is that TAL effectors are under very tight selective pressure and the variable sites in the repeats change rather frequently to adjust to the different target gene in the host. We tested if the plant environment creates a selective pressure on these sites using "imperfect" alternative variants of the PthA4. For this purpose, we have constructed multiple artificial TALs. We constructed one perfect variant and 5 other imperfect variants of TAL effector that target the CsLOB1 binding site similarly to PthA4. It should be noted that PthA4 itself do not contain a perfect CsLOB1 binding site sequence, having two miss matches in sweet orange. We then conducted In planta evolution experiment to evaluate the potential evolution of X. citri. Functional characterization of the citrus canker susceptibility gene CsLOB1. In this objective, we continue to characterize PthA and CsLOB1. PthA, the pathogenicity factor of Xanthomonas citri, directs the expression of the canker susceptibility gene CsLOB1. Subcellular localization analysis of CsLOB1 protein in citrus protoplast revealed that CsLOB1 is primarily localized in the nucleus. We showed that CsLOB1 expression driven by dexamethasone (DEX) in CsLOB1-GR transgenic plants is associated with pustule formation following treatment with DEX. Pustule formation was not observed in DEX-treated wild-type plants and in non-treated CsLOB1-GR transgenic plants. Water soaking is typically associated with symptoms of citrus canker. Weaker water soaking was observed with pustule formation in CsLOB1-GR transgenic plants following DEX treatment. When CsLOB1-GR-transgenic Duncan grapefruit leaves were inoculated with Xcc306ΔpthA4 and treated with DEX, typical canker symptoms, including hypertrophy, hyperplasia and water soaking symptoms, were observed on DEX-treated transgenic plant leaves, but not on mock-treated plants. Twelve citrus genes that are induced by PthA4 are also stimulated by the DEX-induced expression of CsLOB1. As CsLOB1 acts as a transcriptional factor, we identified putative targets of CsLOB1 via bioinformatic and electrophoretic mobility shift assays. Cs2g20600, which encodes a zinc finger C3HC4-type RING finger protein, has been identified to be a direct target of CsLOB1. This study advances our understanding of the function of CsLOB1 and the molecular mechanism of how Xcc causes canker symptoms via CsLOB1.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Ference Cm, Gochez Am, Behlau F, Wang N, Graham Jh, Jones Jb, 2017. Recent Advances in Understanding Xanthomonas citri subsp. citri Pathogenesis and Citrus Canker Disease Management. Mol Plant Pathol.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Duan S, Jia H, Pang Z, Teper D, White F, Jones J, Zhou C, Wang N. 2018 Functional characterization of the citrus canker susceptibility gene CsLOB1. Mol Plant Pathol. doi: 10.1111/mpp.12667
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhou X, Teper D, Andrade MO, Zhang T, Chen S, Song WY, Wang N. 2018 A Phosphorylation Switch on Lon Protease Regulates Bacterial Type III Secretion System in Host. MBio. 23;9(1). pii: e02146-17. doi: 10.1128/mBio.02146-17.