Progress 06/15/17 to 06/14/21
Outputs
Target Audience:Information was shared with scientists and students attending the annual meetings of the Society of Nematologists and the America Society for Plant Biologist, and the Congress of the International Society for Molecular Plant-Microbe Interactions. In addition, information was presented at a public lecture organized by the University California Riverside delivered by first generation faculty including the PI. Changes/Problems:Due to COVID-19 mandated shut down of Riverside county, hence our University, resulted in loss of plant material and the first set of CRISPR tomatoes generated. In addition, the following reduced campus access and level of research, delayed the accomplishing our project goals. It also necessitated to request no cost extension for this grant. Furthermore, the continuous electrical outages on our campus and malfunctioning of greenhouse cooling systems, twice destroyed additional nematode screens in greenhouses resulting in major delays in completing the proposed work. What opportunities for training and professional development has the project provided?This research program provided professional training to five undergraduate students, a graduate student three postdoctoral fellows and a part time research scientist. Weekly group meetings were conducted either discussing research findings or discussing the literature on plant immunity. One-on-one weekly or bimonthly meetings were also conducted with the participants to assist in their scientific proficiency training. All these activities continued via zoom during COVID restrictions. In addition to the pandemic, conference attendance by lab members was limited because of personal family related reasons. How have the results been disseminated to communities of interest?This research was presented at several venues including: 1) University of California ANR Nematology workgroup annual meeting; 2) The Society of Nematologists Annual Meetings in Albuquerque, New Mexico, and in Raleigh, North Carolina; 3) Annual meeting of the Society for Plant Biologists, San Jose, California; 4) Congress of the International Society for Plant-Microbe Interactions, Glasgow, Scotland; and 5) at a public lecture at University of California Riverside. Additionally, results from this work were presented at seminars at: 1) University of California Davis and 2) North Carolina State University, Raleigh. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1. Characterize AT1G61550 and its mutants. Mutations in AT1G61550 (SALK_128729 and SAIL_63_G02) results in enhanced resistance to root-knot nematode (RKN) Meloidogyne incognita. Accordingly, the locus was named ENHANCED RESISTANCE TO NEMATODES (ERN1) and the mutants designated ern1.1 (SALK_128729) and ern1.2 (SAIL_63_G02). Sequence analysis indicated that the ERN1 encodes a G-lectin receptor kinase (G-LecRK) with a G-type lectin domain, a single transmembrane domain, and an intracellular serine/threonine kinase domain. In addition, ERN1 contains additional extracellular domains including: S-locus glycoprotein, an epidermal growth factor-like (EGF), and a plasminogen-apple-nematode (PAN). We performed phylogenetic analysis of Arabidopsis G-LecRKs which classified ERN1 as G-LecRK-VIII.8. Several experiments were carried out to characterize the ern1.1 and ern1.2 mutants. Our initial observations have indicated that both mutants do not exhibit altered plant growth phenotypes. To confirm the plant growth phenotype, mutant and wild-type (WT) seeds were bulked at similar time and seedlings were grown in soil under different temperature (19C or 22C) and photoperiod (16h or 12h daylight) conditions. Measurements of the rosette leaf size showed no statistically significant difference in the growth of the above ground tissues of the mutants compared to the wild-type plants grown under any of the conditions tested. To assess the root growth phenotype, root growth and root capacity were monitored in seedlings grown on nutrient agar plates. No significant difference in root growth or root capacity was observed between the mutants and the wild-type plant roots. The enhanced resistance displayed by the ern1 mutants and lack of obvious root growth phenotypes suggests enhanced immunity in these mutants. We therefore evaluated enhanced immune responses in the mutant and WT roots. Roots were exposed to nematode elicitors such as NemaWater, infective juvenile or RKN egg protein extracts to trigger a synchronized, measurable and efficient response. Defense gene expression by RT-qPCR, MAPK activity by Western blotting, and H202 accumulation by DAB (3,3′-Diaminobenzidine) staining were evaluated. All three defense evaluations, showed that both ern1 mutants have elevated immune responses indicating that ERN1 is a negative regulator of immunity. To confirm the gene causing this mutation, complementation experiments were performed. Two different complementation constructs in binary vectors were developed: 35S::ERN1-His and ERN1pro::ERN1-GFP. Plants transformed with the native promoter construct tagged with GFP were also used for subcellular localization of the protein. Using floral dip technique, we transformed the constructs into the ern1.1 mutant. Screening homozygous T3 plants with RKN, showed that the transgenic plants of either constructs had high levels of nematode infections similar or even higher levels than the susceptible WT Col-0, indicating successful complementation of the mutant phenotype and confirming that AT1G61550 is ERN1. In addition, these complemented lines had reduced levels of flg22-triggered luminol-based ROS burst in leaf tissues compared to the mutants, indicating lower or compromised immune responses. Interestingly, these complemented lines had a bigger rosette diameter and leaf size compared to the WT plants. Immune responses in the complemented plant roots and mutant roots were evaluated by treating with nematode egg extracts or flg22 elicitors and DAB staining. Roots of the ern1.1 and ern1.2 mutants displayed more intense DAB-based brown stain consistent with an enhanced level of immune response, while the complemented lines had similar brown stain intensity as the WT Col-0, consistent with reduced immune response compared to the mutants. Using transient expression in Nicotiana benthamiana as well as the ERN1pro::ERN1-GFP complemented lines, ERN1 was localized to both plasma membrane and the ER confirming its predicted subcellular localization. To explore the role of ERN1 as immune regulator against microbial pathogens, we screened the ern1.1 and ern1.2 mutants with the fungal pathogen Botrytis cinerea and the bacterial pathogen Pseudomonas syringae. Mutants and WT Arabidopsis seedlings were inoculated with P. syringae either by syringe infiltration or floating the seedlings in a bacterial suspension. Using either approach, both ern1.1 and ern1.2 mutants had reduced bacterial titers compared to the WT indicating that ERN1 acts also as negative regulator of immunity to P. syringae. Unlike P. syringae, no difference in fungal infection rate and lesion size were observed on the different plant genotypes indicating differential roles for ERN1 depending on the nature of the pathogen. 2. Characterize the AT1G61550 tomato ortholog(s). To better characterize AT1G61550/ERN1 we identified both Arabidopsis and tomato G-type LecRKs and performed phylogenetic analyses to identified orthologous sequences. We identified 38 Arabidopsis G-LecRKs, seven more than previously reported. We also identified previously uncharacterized 73 members in tomato. Further, using members of G-LecRK from two additional plant species (rice and columbine), we performed phylogenetic analysis and built phylogenetic trees to infer relationships. Based on this thorough analysis, we confirmed the identity of the previously identified tomato orthologs of ERN1. Using the tomato G-LecRK resources we developed, we initially created virus-induced gene silencing (VIGS) gene-specific silencing constructs. The constructs were cloned into tobacco rattle virus vector and introduced into Agrobacterium tumefaciens. Tomatoes were treated with the Agro containing either single gene or co-silenced (for both genes) constructs were used in root-knot nematode assays. Plants silencing with all three constructs resulted in lower levels of nematode infection measured by the number of nematode egg masses. Using the same resources, we developed CRISPR-Cas9 gene-specific constructs targeting deletions in either of the two family members. Those two CRISPR-Cas9 vectors contained sgRNA targeting either one of the two tomato genes. To evaluate the editing efficiency of these constructs, they were used in Rhizobium/Agrobacterium rhyzogenes transformation of tomato cotyledons to generate hairy roots for genotyping. Using PCR, deletions with one of the constructs in the targeted gene was detected in 15% of the transformed hairy roots, with 2/37 roots giving homozygous deletions. The successful construct was used in stable Agrobacterium tumefaciens-based tomato cotyledon transformation. A new CRISPR-Cas9 construct was developed to replace the CRISPR-Cas9 construct that did not yield mutation of the targeted gene. Unfortunately, we lost the initial CRISPR-Cas9 developed tomato plants and had redo the tomato transformation. The second attempt was successful and the CRISPR-Cas9 edited seedlings were used in nematode screens. Regrettably, we lost these plants too for unforeseen electrical problems in our greenhouse. 3. Initiate characterization of the remaining putative PRRs. Mutant seeds for several of the putative PRRs were obtained from various Arabidopsis seed stock centers. Most of the seeds were heterozygous for the mutations and selfing and genotyping was employed to obtained homozygous seeds for these mutants. Homozygous mutant seeds for four of the putative pattern recognition receptors (PRRs) were developed, with two mutant alleles per gene. The two allelic mutants for each of the four putative PRRs were screened on agar plates with M. incognita. Over 30 seedlings per genotypes were inoculated with 100 infective-stage juveniles per plant and screens were repeated once. In both repeats, all mutants had similar levels of nematode infection as their corresponding wild type plants, indicated that none of these putative PRRs are involved in nematode recognition.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Teixeira, M. A., A. Rajewski, O. G. Castaneda, Jiangman He, Amy Litt and I. Kaloshian. 2018. Classification and phylogenetic analyses of the Arabidopsis and tomato G-type lectin receptor kinases. BMC Genomics 19:239
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Kaloshian, I. and M. Teixeira. 2019. Advances in plant-nematode interactions with emphasis on the notorious nematode genus Meloidogyne. Phytopathol. 109:1988-1996. https://doi.org/10.1094/PHYTO-05-19-0163-IA
- Type:
Journal Articles
Status:
Submitted
Year Published:
2021
Citation:
A G-lectin receptor kinase is a negative regulator of Arabidopsis immunity against root-knot nematode Meloidogyne incognita
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Progress 06/15/19 to 06/14/20
Outputs
Target Audience:Scientists and students attending the annual meetings of the Society of Nematologists and the America Society for Plant Biologist, as well as the Congress of the International Society for Molecular Plant-Microbe Interactions. Changes/Problems:Due to COVID-19 related shut down of our University and current reduced level of research, we are facing delays in accomplishing the goals of this projects. No cost extension was requested for this grant and an approval was received. What opportunities for training and professional development has the project provided?This research program provided professional training to one undergraduate student, a graduate student and a postdoctoral fellow. Weekly group meetings were conducted either discussing research findings or the literature on plant immunity. One-on-one weekly or bimonthly meetings were also conducted with the participants to assist in their scientific proficiency training. How have the results been disseminated to communities of interest?This research was presented at the 1) Society of Nematologists Annual Meeting, Raleigh, North Carolina; 2) Annual meeting of the Society for Plant Biologists, Plant Biology 2019, San Jose, California; 3) Congress of the International Society for Plant-Microbe Interactions, Glasgow, Scotland. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Plant parasitic nematodes are microscopic worms causing over $US157 billion annual crop losses worldwide. Root knot nematodes (RKNs) are the most economically important plant nematode parasites worldwide. Chemical control, using nematicides, are the most common mode of control of this group of nematodes in the US. In a handful of major crops, breeding for resistance to these group of parasites exists. While successful to control some nematode species in certain crops, resistance sources have not been identified in a number of crop species and therefore no resources for breeding exists. For those crops with resistance, persistent use of the same resistance source risks creation of virulent nematode populations that are able to overcome the resistance. Current research seeks to identify alternate sustainable approaches to control this important group of parasites. Our work uses gene-editing technology to develop tomato plants that withstand root-knot nematode infections. We are able to do this by removing a negative regulator of plant immunity, a similar approach to immunotherapy used for cancer treatment in human. We have identified a negative immune regulator which is encoded by a lectin receptor-like kinase (LecRK) in Arabidopsis. Mutations of this gene, that eliminate its function, results in plants that are resistant to RKN. These mutants display stronger immune responses than the wild type original plants. Silencing homologous genes in tomato resulted in enhanced resistance to root-knot nematodes. Taken together, gene editing targeted to eliminate this/these gene(s)' function will highly likely work to develop sustainable RKN resistant tomatoes. Goal 1. Characterize AT1G61550 and its mutants. Two AT1G61550 mutant lines (#12 and #16) have been identified exhibiting enhanced resistance to root-knot nematode Meloidogyne incognita. Mutants were analyzed for changes in disease phenotype caused by the bacterial pathogen Pseudomonas syringae. Mutant and wild type Arabidopsis Col-0 seedlings were inoculated P. syringae either by syringe infiltration or floating the seedlings in bacterial suspension. Using either approach, both AT1G61550 (#12 and #16) mutant lines showed reduced bacterial titers compared to the Col-0 wild-type parent indicating AT1G61550 acts also as negative regulator of immunity to P. syringae. The AT1G61550 mutant lines (#12 and #16) were also evaluated for defense gene expression. Mutant and wild-type Arabidopsis Col-0 seedlings were treated overnight with nemawater, water in which RKN infective-stage juveniles are incubated for 24 hours, as well as with RKN egg extracts. Expression of transcription factor MYB51 and cytochrome P450 CYP71A12 transcript levels were significantly upregulated in both #12 and #16 mutant lines compared to the wild-type exposed to either elicitor treatments, indicating that immune responses against RKN are heightened in both mutants. Goal 2. Characterize the AT1G61550 tomato ortholog(s). We had identified two putative tomato orthologs of the AT1G61550 LecRK. We had also identified all members of the tomato LecRK gene family. Using this resource, we developed CRISPR-Cas9 gene-specific constructs targeting deletions in either of the two family members. Those two CRISPR-Cas9 vectors contained sgRNA targeting either one of the two tomato genes. To evaluate the editing efficiency of these constructs, they were used in Rhizobium/Agrobacterium rhyzogenes transformation of tomato cotyledons to generate hairy roots for genotyping. Using PCR, deletions with one of the constructs in the targeted gene was detected in 15% of the transformed hairy roots, with 2/37 roots giving homozygous deletions. The successful construct was used in stable Agrobacterium tumefaciens-based tomato cotyledon transformation. A new CRISPR-Cas9 construct was developed to replace the CRISPR-Cas9 construct that did not yield mutation of the targeted gene. Goal 3. Initiate characterization of the remaining putative PRRs. Nothing to report. Completed previously.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Kaloshian, I. and M. Teixeira. 2019. Advances in plant-nematode interactions with emphasis on the notorious nematode genus Meloidogyne. Phytopathol. 109:1988-1996. https://doi.org/10.1094/PHYTO-05-19-0163-IA
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Progress 06/15/18 to 06/14/19
Outputs
Target Audience:Scientists and students interested in immunity to nematodes, attending the Annual meeting of the Society of Nematologists, are among the target audience for this period. Additional audience were the riverside public at a lecture organized by the University delivered by first generation faculty. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This research program provided professional training to one undergraduate student, a graduate student and a postdoctoral fellow. Weekly group meetings were conducted either discussing research findings or the literature on plant immunity. One-on-one weekly or bimonthly meetings were also conducted with the participants to assist in their scientific proficiency training. How have the results been disseminated to communities of interest?This research was presented at the Society of Nematologists Annual Meeting in Albuquerque, New Mexico, and at a public lecture at University of California Riverside. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Plant parasitic nematodes are microscopic worms causing over $US157 billion annual crop losses worldwide. Among these nematodes, the most economically important groups are the root-knot nematodes and cyst nematodes. The main measure used to control these parasites is soil fumigation by methyl bromide or chemical pesticides. In a few major crops, breeding for resistance to these parasites exists. While successful to control some nematode species in certain crops, resistance sources have not been identified in a number of plant species and therefore no resources for breeding exists. In addition, resistance in a number of crops have been overcome by virulent nematode populations. Therefore, an alternate approach is necessary to control this important group of important parasites. Our work uses gene-editing technology to develop tomato plants that withstand root-knot nematode infections. We are able to do this by removing a negative regulator of plant immunity, a similar approach to immunotherapy used for cancer treatment in human. This regulator is encoded by a lectin receptor-like kinase (LecRK) in Arabidopsis. Mutations of this gene that eliminate its function, results in plants that are resistant to the nematode. These mutants display stronger immune responses than the wild type original plants. Silencing homologous genes in tomato resulted in enhanced resistance to root-knot nematodes. Taken together, gene editing targeted to eliminate this/these gene(s)' function will highly likely work to develop root-knot nematode resistant tomatoes. Goal 1. Characterize AT1G61550 and its mutants. Two AT1G61550 null mutant lines (#12 and #16) exhibiting enhanced resistance to root-knot nematode Meloidogyne incognita are used as controls in most of the described experiments. We used mutant line 12 in complementation experiments with three different promoter constructs: the gene native promoter, Cauliflower Mosaic Virus 35S overexpression promoter or the Ubiquitin 10 (UBQ10) overexpression promoter that is expressed at high level in roots. Homozygous T3 plants of all thee constructs had high levels of nematode infections similar or even higher levels than the susceptible wild-type Col-0, indicating successful complementation of the mutant phenotype. In addition, these complemented lines had reduced levels of flg-22-triggered luminol-based ROS burst in leaf tissues indicating lower or compromised immune responses. Interestingly, these complemented lines had a bigger rosette diameter and leaf size compared to Col-0. To characterize the immune responses in roots against nematodes, we used nematode egg extract as elicitor to trigger a synchronized, measurable and efficient response. We tested presence of H202 in roots upon elicitor treatment using DAB (3,3′-Diaminobenzidine) staining. We first tested mutants #12 and #16 and showed that H202 roots of both mutants stained more intense brown compared to Col-0 upon either nematode egg extract or flg22 treatments. In contrast, complemented lines had similar DAB-based brown stain intensity as Col-0, consistent with a reduced level of immune response. Goal 2. Characterize the AT1G61550 tomato ortholog(s). We had identified two putative tomato orthologs of the AT1G61550 LecRK. We had also identified all members of the tomato LecRK gene family. Using this resource, we developed gene-specific silencing constructs. The constructs were cloned into tobacco rattle virus vector and using in virus-induced gene silencing introduced by Agrobacterium tumefaciens. Tomatoes silenced for either gene or co-silenced (for both genes) were used in root-knot nematode assays. Plants silencing with all three constructs resulted in lower levels of nematode infection measured by the number of nematode egg masses. Goal 3. Initiate characterization of the remaining putative PRRs. Homozygous mutant seeds for four of the putative pattern recognition receptors (PRRs) were developed, with two mutant alleles per gene. The two allelic mutants for each of the four putative PRRs were screened with root-knot nematode. All mutants had similar levels of nematode infection as their corresponding wild type plants, indicated that none of these putative PRRs are involved in nematode recognition.
Publications
|
Progress 06/15/17 to 06/14/18
Outputs
Target Audience:This work provided hands on summer research training for two undergraduate students: a female undergraduate minority/hispanic student transferring to our University from a community college and a female UCR student majoring in Biology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This research program provided professional training for two undergraduate students, a graduate student and a postdoctoral fellow. Weekly group meetings were conducted either discussing research findings or discussing the literature on plant immunity. One-on-one weekly or bimonthly meetings were also conducted with the participants to assist in their scientific proficiency training. How have the results been disseminated to communities of interest?A presentation on this research was made at the University of California ANR Nematology workgroup's annual meeting in March 2018. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Plant parasitic nematodes are mostly soil dwelling microscopic worms responsible for over $US157 billion annual crop losses worldwide. Among these nematodes, the most economically important groups are the root-knot nematodes and cyst nematodes. Until recently, the main approach to control these pests in the developing world, including the US, was with fumigation using methyl bromide or by chemical pesticides. In addition to chemical control, breeding for resistance to this group of nematodes is carried out in a few major crops. While successful to control some nematodes, resistance breeding is not possible for many crops as resistance loci to these pests have not been identified or are difficult to incorporate into cultivated plant species. In addition, resistance is typically conferred by a single gene which is frequently overcome by the appearance of virulent nematode populations in the field. Therefore, an alternate approach is necessary to control this important group of pests. Our proposal uses gene-editing technology to develop tomato plants that withstand root-knot nematode infections. We are able to do this based on identification of a mutation in a G-type lectin receptor-like kinase (LecRK) in Arabidopsis that results in enhanced resistance to this nematode. Understanding the mechanism of this enhanced resistance and mirroring the mutation in tomato, using gene editing, is the goal of our proposed research. Goal 1. Characterize AT1G61550 and its mutants. Two AT1G61550 mutant lines (#12 and #16) have been identified exhibiting enhanced resistance to root-knot nematode Meloidogyne incognita. Several experiments have been carried out to characterize these two mutant lines (#12 and #16). Our initial observations have indicated that both mutants do not exhibit altered plant growth phenotypes. To confirm the plant growth phenotype, seedlings were grown in soil under different temperatures (19C or 22C) and phtoperiod (16h or 12 h daylight) conditions. Measurements of the rosette leaf size showed no statistically significant difference in the growth of the above ground tissues of the mutants compared to the wild-type plants grown under any of the conditions tested. To assess the root growth phenotype, root growth and root capacity was monitored in seedlings grown on nutrient agar plates. No significant difference in root growth or root capacity was observed between the mutants and the wild-type plant roots. Mutants were also analyzed for changes in disease phenotype caused by the fungal pathogen Botrytis cinerea. Mutant and wild type seedlings inoculated with B. cinerea spores had similar infection rate and lesion size indicating no role for this LecRK in Botrytis defense. To confirm the gene causing this mutation, complementation experiments were initiated. Three different complementation constructs in binary vectors have been developed. The difference among these constructs is the nature of the promoters which are: the gene native promoter, Cauliflower Mosaic Virus 35S overexpression promoter or the Ubiquitin 10 (UBQ10) overexpression promoter that is expressed at high level in roots. Both native and UBQ10 promoter constructs have GFP tags to assist with subcellular localization. The 35S construct has a His tag. Both GFP and His tags will be used for successful transgene protein expression analysis. Using floral dip technique, we transformed all three constructs into Arabidopsis mutant #12. We have obtained transgenic plants for all three constructs and are in the process of selecting single insertion T3 transgenes for further analysis. Goal 2. Characterize the AT1G61550 tomato ortholog(s). We had initially identified two putative tomato orthologs of the AT1G61550 LecRK. To confirm this orthologous relationship, we identified both Arabidopsis and tomato G-type LecRKs and performed phylogenetic analyses to identified orthologous sequences. We identified 38 Arabidopsis G-LecRKs, seven more than previously reported. We also identified previously uncharacterized 73 members in tomato. Further, using members of G-LecRK from two additional plant species (rice and columbine), we performed phylogenetic analysis and built phylogenetic trees to infer relationships. Based on this thorough analysis, we confirmed the identity of the previously identified tomato orthologs of the AT1G61550 LecRK. Goal 3. Initiate characterization of the remaining putative PRRs. Mutant seeds for several of the putative PRRs have been obtained from various Arabidopsis seed stock centers. Genotyping these mutants indicated that the majority of the mutants were heterozygous for the mutations. We are in the process of generating homozygous bulk seeds for these mutants.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Teixeira, M. A., A. Rajewski, O. G. Castaneda, Jiangman He, Amy Litt and I. Kaloshian. 2018. Classification and phylogenetic analyses of the Arabidopsis and tomato G-type lectin receptor kinases. BMC Genomics 19:239
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