GENETIC ANALYSIS AND RNA MARKER DEVELOPMENT FOR FOV4 RESISTANCE BREEDING IN COTTON

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1026954
• Grant No.: 2019-67029-35289
• Cumulative Award Amt.: $201,261.28
• Proposal No.: 2021-08632
• Project Start Date: Feb 15, 2021
• Project End Date: Aug 14, 2022
• Grant Year: 2021
• Program Code: [A1701]- Critical Agricultural Research and Extension: CARE

Recipient Organization
TEXAS TECH UNIVERSITY
(N/A)
LUBBOCK,TX 79409

Performing Department
Plant and Soil Science

Non Technical Summary
Fusarium wilt, caused by [Fusarium oxysporum f. sp. vasinfectum (FOV)], is a serious economic disease of cotton (Gossypium hirsutum) in the U.S. and many other countries. FOV is a soil and seed borne pathogen surviving for long periods without a host and cannot be eradicated once established. Recently, new extraordinarily pathogenic FOV isolates have appeared on cotton in the U.S. FOV race 4 (FOV4) has been identified in California soils in 2003 and since then, has spread throughout California's San Joaquin Valley. FOV4 now has been found around El Paso, Texas now threatening Upland cotton production in Texas, the largest cotton-producing state, and across the contiguous U.S. belt. Furthermore, greenhouse pathogenicity tests show FOV4 severity increases in the presence of root knot nematode (RKN) (Meloidogyne incognita) and FOV race 1 (FOV1) pathogens. The rapid transmission of FOV4 across the U.S. makes the proposed work of utmost urgency, leading to short- to medium-term application of results providing solutions that will be implemented within two years of the project conclusion that aids stakeholders. Host-plant resistance is the only cost-effective solution, seriously impeded by Upland cotton's narrow genetic base. Consequently, conventional breeding has introgressed genes for tolerance/resistance to FOV4, providing a diagnostic basis to understand the genetic and genomic basis for FOV4 tolerance/resistance mechanisms in cotton. The hypothesis is that the cotton resistance mechanism to FOV4 is genetically based and will be determined by identifying genetic polymorphisms and gene expression characteristics relating to defense through the well documented capability of RNA sequencing (RNA seq), expediting breeding for FOV4 resistance in Upland cotton from a diversity of Gossypium sources. Experimental reactions are analyzed in the project, spanning eight cotton genotypes, selected based on their known FOV4, FOV1 and RKN resistance/tolerance/susceptible profiles, their current use as whole genome reference sequence genotypes, and as parents of recombinant inbred line (RIL) genetic mapping populations. These genotypes include Barbren-713-32-38 (FOV4 susceptible) and a backcrossed-derived isoline (BC5S2, FOV4 resistant introgressed from G. arboreum), both FOV1 and RKN resistant; Pima S-7 and Pima 379 (Pima types highly susceptible to FOV4, tolerant to FOV1); Acala NemX and TM1 and UH001 (tolerant/resistant to FOV4, susceptible to FOV1 and for NemX RKN resistant); Pima S-6 (tolerant to FOV4, susceptible to FOV1 and RKN). Pima S-7, Pima 379, TM1 and NemX are RIL population parents. The proposed project is facilitated by the TM1 primary G. hirsutum genome reference. Other employed genomes include G. barbadense, 243 additional re-sequenced cotton genomes, FOV1 and RKN. The in-house RNA-seq pipeline analyzes cotton responses to soil-infestation in series of inoculations as the Project Narrative outlines. As demonstrated in related studies RNA-seq analyses clarify, genetically, knowledge of defense (and susceptibility) occurring in a disease matrix made through morphological studies. Expressed FOV and RKN genes in the different pathogen infected reactions are also identified, leading to the identification of pathogen effectors that can be used in cotton FOV defense receptor identification. In conjunction with SNP map data, key defense/tolerance/susceptibility genes are identified, subjected to functional validation using in-house transgenic procedures (overexpression, RNA interference (RNAi), virus induced gene silencing (VIGS) and CRISPR/Cas9). Validated defense genes are used in response to develop RNA based markers to aid FOV4 resistance breeding, making tools available to scientists, extension, stakeholders and public.

Animal Health Component

10%

Research Effort Categories

Basic

85%

Applied

10%

Developmental

5%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1710	1040	50%
201	1710	1080	50%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1710 - Upland cotton;

Field Of Science
1040 - Molecular biology; 1080 - Genetics;

Keywords

cotton

fusarium wilt resistance

root knot nemat

rna-seq analysis.

Goals / Objectives
Fusarium wilt caused by [Fusarium oxysporum f. sp. vasinfectum (FOV)] is a soil and seed borne pathogen surviving in soil for long periods without a host. It cannot be eradicated once established. In the last decade and a half, new isolates of Fusarium that are extraordinarily pathogenic on cotton have appeared in the U.S. In 2003 FOV race 4 (FOV4) was identified in California soils. Since then, FOV4 has spread throughout California's San Joaquin Valley. In 2017, FOV4 has also been found around El Paso, Texas now threatening Upland cotton production in Texas, the largest cotton-producing state, and across the contiguous U.S. belt. Greenhouse pathogenicity tests show FOV4 severity increases in the presence of root knot nematode (RKN) (Meloidogyne incognita) and FOV race 1 (FOV1) pathogens. Host-plant resistance is the only cost-effective solution, seriously impeded by Upland cotton's narrow genetic base. In this proposed research study, the cotton resistance mechanism to FOV will be determined by identifying genetic polymorphisms and gene expression characteristics relating to plant defense mechanisms for FOV and molecular tools will be developed expediting breeding for FOV4 resistance in Upland cotton from a diversity of Gossypium sources. Three research objectives are proposed for this study:1. Determine the susceptibility and resistance responses of eight cotton genotypes to FOV race 1 (FOV1), FOV4 and rootknot nematode (RKN) at the molecular level. To assess the susceptibility and resistant response, selected genotypes will be subjected to a series of pathogenicity evaluations and pathogen combinations of soil artificial inoculations with none or control, FOV1, FOV4, RKN, FOV1+RKN, FOV4+RKN. From these artificial inoculations, root of plant tissue will be obtained for RNA-seq experiments. The expert laboratories include Roberts, Bell, Liu, Ulloa.2. Identify candidate resistance genes from transcriptome profiles of infected cotton tissues and single nucleotide polymorphism (SNP) or biomarkers. RNA samples from tissue of exposed/infected cotton plants with the pathogen and uninfected or control will be sequenced using the next generation technology (RNA-seq). Gene expression data will be analyzed to dissect molecular regulatory networks and association with single nucleotide polymorphisms (SNPs) involved with plant defense mechanisms and pathogenicity that will help identify candidate defense/resistance genes. RNA-seq analysis will be carried out using an in-house pipeline in response to soil-infestation of series of inoculations. The expert laboratories include Lopez-Arredondo and Herrera-Estrella at Texas Tech University.3. Initiate candidate gene functional testing, identifying major candidate genes. Efforts to validate the identified candidate defense genes will be initiated. Validation of defense genes will be carried out using in-house-developed transgenic to provide tools to scientists. The expert laboratories include Lopez-Arredondo and Herrera-Estrella at Texas Tech University.

Project Methods
The project is executed in three phases:Phase I. Eight cotton genotypes that have already been identified by the collaborators to experience defined reactions to FOV4, FOV1 and RKN, are evaluated to verify phenotype and set up experimental conditions. The outcomes of those infections are susceptibility, resistance or tolerance. Labs of collaborators will share and use similar known/confirmed FOV1 and FOV4 isolates and RKN strain, and will share seed sources to perform greenhouse evaluations. The whole team will work together to stablish and optimize protocols to produce inoculum and to inoculate cotton plants for infection experiments.Fusarium wilt assays. Seeds are germinated for one week, seedlings are uprooted, the roots are rinsed with tap water, trimmed to 2-3 cm length, and immediately dipped for 3 min into a suspension of FOV inoculum. Seedlings are then transplanted into steam-sterilized substrate and pots are arranged in a randomized complete block design in the greenhouse. Wilt symptoms start to show at 7-9 days after inoculation (DAI), and disease severity is measured periodically. Individual plants are rated for disease severity based on the following index: 0=no symptoms; 1=epinasty and slight dwarfing; 2=1 to 30% of chlorotic leaves; 3=31 to 80% of chlorotic leaves and severe stunting; 4=81 to 100% of chlorotic leaves; and 5=plant death. Multiple evaluations of disease progress are combined into a single value by using the area under the disease progress stairs and calculated according a reported formula. The stems are cut longitudinally and evaluated for vascular discoloration at 23 DAI. An alternative direct-seeded method is also proposed for this study for FOV inoculation that can be used in combination with RKN artificial inoculation. This method involves the artificial inoculation of the substrate which is kept covered and moistened for 5 to 6 days, and every day the inoculated soil is mixed to homogenize the distribution of the inoculum. The soil is then placed in pots and seed is planted. Disease infection is rated after susceptible checks show strong foliar symptoms. Data is analyzed using ANOVA.RKN Assay. Three-week-old seedlings are inoculated with RKN. After inoculation, pots are drip-irrigated to maintain plant growth. Greenhouse conditions are maintained daily between 28-35oC and 24oC at night. The experiment is arranged in a randomized complete block design with four replications. Inoculated plants are evaluated for response to nematode using a 0-10 root-gall rating scale as follows: 0=no galls, 1=few small galls, 2=<10% of roots infected, 3=10-30% of roots infected, 4=31-40% of roots infected, 5=51-60% of roots infected, 6=61-70% of roots infected, 7=71-80% of roots infected, 8 = 81-100% of roots infected, 9=all roots severely knotted and plant usually dying, 10=all roots severely knotted with diminished root system and plant usually dead. For further assessment of the resistance/susceptibility response to RKN infection, the number of nematode eggs per gram of fresh root is calculated. Nematode eggs are extracted from the roots and counted using a stereo-microscope. Data is analyzed using ANOVA. FOV and RKN infection experiments will be performed through years 1 and 2.Phase II. Root tissue is harvested for total RNA isolation. Transcriptome profiles are generated and bioinformatics analyses to identify genes that relate to resistance, susceptibility, and/or tolerance aided by the available cotton, FOV1 and RKN genomes are conducted.Root collection for RNA isolation for RNAseq. Root samples from each genotype/treatment are collected in 3 replicates, frozen and ground in liquid N2, and total RNA is isolated following reported protocols. Tissue is collected at two times during the experiments, at a) 3 and b) 8 DAI. After isolation, the yield and purity of RNA are analyzed with a ND-1000 Spectrophotometer. Only RNA samples with 1.8 - 2.2 ratio of absorbance 260/280 nm are kept and used for cDNA library preparation. High quality RNA samples are expected. RNA sequencing will be performed through years 2 and 3 of this project.Bioinformatic analyses. Total RNA is used for library preparation and sequencing. mRNA libraries will be sequenced by NGS technologies using Illumina NovaSeq system to generate paired-end reads from coding RNA sequences for further manipulation. Data will be stored in the High-Performance Computing Center at Texas Tech University and in our own server at the Experimental Science Building II. A classic RNA-seq pipeline will be followed for the differential expression analysis: quality of raw reads will be first checked using FastQC and low-quality reads and adapter sequences are removed using Trimmomatic. The clean reads are analyzed and aligned to the publicly available cotton genomes at NCBI using HISAT v2.0.5. Transcripts will be mapped and indexed to the reference genome using Kallisto or Salmon software and gene annotation is made using Blast2GO. Then, differential expression analyses calculated based on the fold change between the control conditions and each treatment condition, of the different conditions are performed. Bioinformatic analysis will be facilitated by genome sequences already available for G. hirsutum and G. barbadense (our lab). Genome sequences of FOV1 and RKN are also available. These datasets alone will significantly inform on the transcriptomic responses that the different cotton genotypes are deploying to cope with the respective stress input.Gene prioritization. Bioinformatic analysis will be directed to identify gene expression occurring within and between the cotton genotypes under investigation. Expression of a set of genes will be common to all treatments. Some gene's activity occurring during compatible, incompatible or tolerant interactions will be unique, revealing specific alterations in expression or gene sequence that underlie the studied process. Regulatory networks will be constructed to identify central nodes that could constitute important components of the resistance mechanisms. Likewise, the analysis will allow us to identify potential defense genes whose expression relates to a particular type of infection and/or combination of infections. These genes would be considered to have the highest level of correlation to a defense reaction and of highest importance in selecting candidates for functional test. Bioinformatic analysis will be performed during year 3 of this project.Phase III. Efforts to initiate biological validation of the selected candidate resistance genes based on the provided hierarchical criteria, are performed during this phase. The functional tests lead to the identification of resistance and susceptibility genes that can be used in breeding programs.Based on selected genes, gene constructs will be designed and chemically synthesized (GenScript) or PCR amplified to proceed with biological validation. As an initial attempt, hairy roots will be utilized; HR screening rapidly screens candidate genes for defense roles toward root and stem pathogens. Cotton shoots from cut plants are transferred to MS media containing A. rhizogenes harboring DNA constructs. Transgenic root primordia are evident 5 days after planting using eGFP confirmed by qPCR validation. The generation of cotton composite plants with transformed roots can be considered. From these experiments, clear insights about the role of the selected genes on the infection mechanisms will be generated, opening opportunities to initiate further and more complex experiments and to conduct stable genetic transformation efforts to introduce these traits into highly productive cotton varieties already in the market, thus, in benefit of cotton growers. Biological validation of candidate genes will be carried out during year 3 of this project.

Progress 02/15/21 to 08/14/22

Outputs
Target Audience:Diseases caused byRKN and Fusariumthreatencotton cultivation. The highly virulent Fusarium strain FOV4, ischallengingthe current management schemes and cotton farmers response capacity, making the need of new and more effective strategies to develop resistant cotton germplasm a matter of urgency. Therefore, our target audienceis academic and scientific community working on plant pathology and biology in general, and cotton growers and breeders. The efforts focused onexperiments and data analyses performed in the lab, greenhouse and field for the project pertaining to the funded proposal, and dissemination of results through research articles and local and international meetings. Efforts also included the training of a postdoctoral fellow and a special topic class in which the research results were presented as part of transcriptomicphenotyping of plants. Results were also presented to researchers and breeders in the annual Cotton Inc meeting. Changes/Problems: Progress was delayed due to the transfer process of this project in 2021 from Dr.Vincent Klinkto Dr. Lopez-Arredondo who became the new PIfor the project, and adding a new Co-PI, Dr. Herrera-Estrella. During the first year of this project,experimental conditions of pathogenicity tests were stablished, but generation of cotton root samples and RNA-seq analysis were not conducted until Dr. Lopez-Arredondondo joined the project.Restrictions and limitations imposed by the pandemic also slightly affected setting up final experiments to generate root samples, and RNA sequencing data delivery by the outsourced company due to personnel restrictions which slightly delayed bioinformatic analyses. This delay impacted a more comprehensivebiological validation of the resistancecandidate genes identified, related to Objective 3. Genetic transformation of cotton is quite laborius andrequires highy skilled hands, and the generation of the first progeny of transformed plants can take up to 18months. Therefore, stable genetic transformation using the different geneconstructs we designed is something unfeasible for the duration of this project. Our strategy was refocused to implementdifferent bioinformatic analysis to study DNA and protein sequences of cotton candidate genes in comparison to their orthologues inArabidopsis and other crops, and to includeArabidopsis as the model plant to gain insights on the biological function of the resistance candidate genes. This allowed us to expand ourknowledge on the RKN and FOV4 resistant responses and design strategies for the use of these genes for genetic improvement of cotton. What opportunities for training and professional development has the project provided?This project has provided opportunities for training and professional development at the laboratories of all PI and Co-PIs. Opportunities in the Ulloa, Roberts and Liu labs were mainly for technicians/postdoctoral associates and graduate students to perform seed multiplication in the field and infection trials in the greenhouse and field. These activities were crucial to generate the root samples from the different cotton entries subjected to the infection treatments that were sent to Lopez-Arredondo/Herrera- Estrella lab for RNA isolation, RNA sequencing, and bioinformatic analysis performed in this project. Opportunities in Lopez-Arredondo/Herrera-Estrella labs were for postdoctoral associates and graduatestudents and included growth of cotton under greenhouse, delinting, surface disinfection and in vitro germination of seeds, set up of liquid chromatography (UHPLC) coupled to mass spectrometry protocols to analyze plant hormone levels and metabolites in cotton roots, RNA isolation, RNA quality assessment, qRT-PCR, RNA-seqbioinformatic analyses, and design and generation of gene constructs for gene overexpression and modulationusing uORFs. These training opportunities were crucial for professional development for publishing results and deliver presentations at meetings and ultimately securing jobs.Students and postdoctoral fellows were also trained in the use of HPLC-MS metabolomic analyis of resistant and susceptible plants infected with FOV4.Skills,capacities andresources developed by the personnel involved in this projectare opening new avenues to study cotton diseases followinga more holistic approach to understand cotton tolerance and sussceptibility responses, and therefore, be able to design more effective breeding strategies. How have the results been disseminated to communities of interest?Diseases casued by RKN and Fusarium are of themajorimportance forcotton cultivation. Therefore, PI,Co-PIs, postdocs and gradate students have actively disseminated themain findings generated in this project.The PI and CoPIsgave several lectures about cotton genomics and transcriptomics, Fusarium and root-knot nematode infection and resistance in cotton to different audiences,including:Beltwide Cotton Conference 2022, Annual review of the Texas State Support Committee meeting 2021 and 2022,CABANA Food Security Knowledge Exchange Meeting 2022,USDA-ARS &TTU Research Spotlight,2022, and 1st Symposium of the Institute of Genomics for Crop Abiotic Stress Tolerance (IGCAST), TTU,2022. Students and postdocs presented scientific posters also. Moreover, attendance and oral presentations have been scheduled at theBeltwide Cotton Conference in January,2023. We have also published part of ourfindings generated in this project in one manuscript; one manuscript iscurrently under review and one more is in preparation. Thus, results have been disseminated to communities of interest including the academic and scientific community working on plant pathology and biology in general, and cotton growers and breeders. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact statement. RNA-seq analysis of infected cotton entries showing different levels of resistance/susceptibility to both RKN and FOV at different time-points post infection allowed us to generate a breakthrough understanding of the plant defense mechanisms for both pathogens. We identified a series of key genes involved in the resistance mechanisms to RKN and FOV4 that are linked to previously identified QTLs involved with the resistance response. These genes can be used to speed up resistance breeding efforts. Our results provide a novel view to understand the genetic and genomic basis for RKN and FOV tolerance/resistance mechanisms in cotton. Objective 1.Pathogenicity evaluations.Wedetermined thesusceptibility and resistance responses to FOV race 1 (FOV1), FOV4 and root-knot nematode (RKN) of more than 10 cotton genotypes, under both greenhouse and field conditions.Two sets of calibration tests were run to determine the optimal artificial inoculation protocol, and the best times of harvesting root for RNA isolation. For inoculated tests, cotton seeds were germinated in seedling trays and then transplanted into greenhouse pots for inoculation with RKN, FOV (1 or 4) or water controls in various combinations. Field evaluations confirmed the greenhouse studies, showing that entries Pima S-7, Pima 3-79 and Stoneville 474 are susceptible, NemX tolerant, and Pima S-6 and PSSU77 resistant to FOV4. Experimental conditions for disease assay were optimized to effectively express symptoms caused by both separate and combined infection treatments (RKN, FOV1, FOV4, FOV1+RKN, and FOV4+RKN), thus enabling a comparative transcriptomic analysis of all treatments under a single experimental protocol. To get insights into the molecular mechanisms and the genetic diversity that underlie resistance and susceptibility responses to both pathogens, final infection experiments were performed with only six cotton entries showing different levels of resistance/susceptibility, i.e., threeG. hirsutum (Gh,Upland): TM-1, NemX, PSSU77, and threeG. barbadense (Gb,Pima): Pima S-6, Pima S-7, Pima 3-79, under all treatments and at two time-points, two and eight-days post infection, with four replicates each. In total, 288 cotton root samples were generated at the collaborator´s laboratories (Drs. Liu and Roberts), which were sent to Dr. Lopez-Arredondo laboratories at Texas Tech. Objective 2.RNA-seq analysis and resistance candidate genes.Frozen root tissue from each treatment/genotype condition was utilized for RNA-sequencing with the Illumina NovaSeq PE150 platform. We determined differentially expressed genes (DEGs) in response to the pathogen combination treatments across all genotypes tested. We detected an important transcriptional response in bothGhandGbentries in response to RKN and RKN/FOV combined treatments. No differential gene expression in response to FOV1 and FOV4 treatments inGhwas detected. We found a large number of DEGs shared for FOV1+RKN, FOV4+RKN and RKN only infections, and sets of genes that were specifically upregulated in response to the combined treatments only, indicating a specific response to FOV in the presence of RKN. Suggesting that RKN facilitates FOV1 and FOV4 infection which trigger the regulation of these additional sets of genes. Gene Ontology (GO) enrichment analysis of these sets of upregulated genes helped us to determine the biological processes activated in response to these multiple infections. In the case of RKN treatment, GO categories related tocell cycleandhormonal regulation pathwayswere activated in bothGhandGb. This is consistent with previous reports showing that nematodes induce the cell cycle and manipulate phytohormone pathways for feeding site formation. In the case of RKN+FOV1 and RKN+FOV4, enriched GO categories were related todefense response to fungusandresponse to fungus and chitin binding, indicating that FOV infection was successful and corroborated our hypothesis that RKN facilitates infection by both FOV races. These findings are relevant because FOV1 field symptoms on Upland cotton have been associated with co-infestation with RKN, and our results showed that there is an additive or synergistic effect on cotton damage between FOV4 and RKN. Additional RNA-seq analysis of three RKN-infectedGhcotton entries SJ-2, WMJJ and NemX that present low, intermediate, and high levels of resistance to RKN, respectively, at 23 days post infection, provided more insights on RKN-resistance mechanisms. We found that RKN-resistance is determined by a constitutive transcriptional priming state of defense in the roots NemX. NemX responses are enriched in jasmonic and salicylic acid-related genes, two key phytohormones in plant defense responses. These genes included cotton genes coding for disease resistance and receptor proteins linked to RKN-resistance and perception in plants. Interestingly, we identified twoGhortholog genes coding for NBS-LRR proteins that have enhanced expression in NemX that mapped to a previously identified RKN-resistance QTL in cotton. We reported these findings in a research article published in Frontiers in Plant Science (Ojeda-Rivera et al., 2022, 13;13:85831). These studies, allowed us to identify genes that can be used to speed up breeding efforts for RKN resistance. Because the transcriptional response to FOV obtained inGbentries was very discrete at early time-points, we performed RNA-seq analysis to study FOV4 infection only in Pima cotton entries at 28 days post infection. We discovered different transcriptional responses between susceptible and resistantGbcultivars to FOV4 infection. As expected, the susceptible entries (P3-79 and PS-7) presented similar transcriptional profiles in response to FOV4. We found that there are less DEGs in FOV4-resistant Pima S-6 in comparison to the susceptible genotypes, and its datasets cluster is more related to the transcriptional profiles of uninoculated control plants. Comparative gene set analysis suggests that the response is largely genotype specific, but categories related tochitin recognitionare common in P3-79 and PS-7 and are not present in resistant PS-6. Most enriched PS-6-specific GO categories are related to genes involved in azelaic acid signaling, a molecule that primes plant immune-responses.AZELAIC ACID INDUCED (AZI)genes, and their closest paralogsDIR1andEARLI1,encode proteins that are transported to the phloem and activate priming of immune responses which results in stronger activation defense responses when exposed to biotic stimulus. Some of the genes specifically expressed in Pima S-6 are indeed close orthologs of azelaic-acid signaling related Arabidopsis proteins AZI1, DIR1 and EARLI1. Objective 3. Initiate gene validation.We selected a set of resistance candidate genes useful to engineer plant disease resistance from our NemX and Pima S-6 transcriptomic data and initiated their functional validation using Arabidopsis as the model plant. These genes were selected mainly on the basis of being more expressed in the resistant entries and related to plant disease immune responses, particularly those located in the cotton QTLs regions associated with RKN and FOV4 resistance.At the time this report was submitted, production of Arabidopsis transgenic seeds was in process and the detailed characterization of the lines will continue. We also initiated the design of molecular biology strategies to test candidate gene alleles from the identified genomic regions of NemX and Pima S-6 in susceptible cotton entries using a self-regulated endogenous system based on Open Reading Frames (uORF). The work reported here wasgreatly facilitated by a collaborative effort of our team for sequencing the genome of Pima-S6 and NemX, which was partially supported by this project. To date the Pima-S6 genome has been completed and a manuscript reporting a reference, chromosome level Pima S-6 genome is under review in BMC Genomics.

Publications

• Type: Journal Articles Status: Other Year Published: 2022 Citation: Ojeda-Rivera JO, Ulloa M, Roberts PA, Kottapalli P, Wang C, N�jera-Gonz�lez HR, Herrera-Estrella L, Lopez-Arredondo D. Transcriptional landscape of the response and resistant to Fusarium vasinfectum in Pima cotton. In preparation.
• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Ch�vez Montes RA, Mauricio Ulloa M, Biniashvili T, Zackay A, Kfir N, Lopez-Arredondo D, Estrella-Herrera L. (2022). Assembly and annotation of the Gossypium barbadense L. 'Pima-S6' genome raise questions about the chromosome structure and gene content of Gossypium barbadense genomes. Under review in BMC Genomics.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Han J, Lopez-Arredondo D, Yu G, Wang Y, Wang B, Wall SB, Zhang X, Fang H, Barrag�n-Rosillo AC, Pan X, Jiang Y, Chen J, Zhang H, Zhou BL, Herrera-Estrella L, Zhang B, Wang K. Genome-wide chromatin accessibility analysis unveils open chromatin convergent evolution during polyploidization in cotton. Proc Natl Acad Sci U S A. 2022 Nov;119(44):e2209743119. doi: 10.1073/pnas.2209743119. Epub 2022 Oct 24. PMID: 36279429; PMCID: PMC9636936.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Ojeda-Rivera JO, Alejo-Jacuinde G, N�jera-Gonz�lez HR, L�pez-Arredondo D. Prospects of genetics and breeding for low-phosphate tolerance: an integrated approach from soil to cell. Theor Appl Genet. 2022 Nov;135(11):4125-4150. doi: 10.1007/s00122-022-04095-y. Epub 2022 May 7. PMID: 35524816; PMCID: PMC9729153.

Progress 02/15/21 to 02/14/22

Outputs
Target Audience:Target audiences were the academic and scientific community working on plant pathology and biology in general, and cotton growers and breeders.The efforts were the experiments and data analyses performed in the lab, greenhouse and field for the project pertaining to the funded proposal, and dissemination of results throughresearch articleand local and international meetings. Changes/Problems:Progress was delayed at the beginning of this reporting period due to the transfer process and Dr. Lopez-Arredondo taking on this project as the new PI. Restrictions and limitations imposed by the pandemic also slightly affected setting up final experiments to generate root samples, and RNA sequencing data delivery by the outsourced company due to personnel restrictions which slightly delayed bioinformatic analyses. What opportunities for training and professional development has the project provided?This project has provided opportunities for training and professional development at the laboratories of all PI and Co-PIs. Opportunities in the Ulloa, Roberts and Liu labs were mainly for technicians/postdoctoral associatesand studentsto perform seed multiplication in the field and infection trials in the greenhouse. These activities were crucial to generate the root samples from the different cotton entries subjected to the infection treatments that were sent to Lopez-Arredondo/Herrera-Estrella lab for RNA isolation, RNA sequencing, and bioinformatic analysis performed during this period. Opportunities in Lopez-Arredondo/Herrera-Estrella labs were for postdoctoral associates and students and included cotton seed harvesting, delinting, surface disinfection and in vitro germination, set up of liquid chromatography (UHPLC) coupled to mass spectrometry protocols to analyze plant hormone levels in cotton, RNA isolation, RNA quality assessment, and RNA-seq bioinformatic analyses. These training opportunities were crucial for professional development for publishing results and deliver presentations at meetings and ultimately securing jobs. How have the results been disseminated to communities of interest?The PIs gave several lectures about cotton genomics and transcriptomics, Fusarium and root-knot nematode infection and resistance in cotton to different audiences in the reporting period, including:2022 Beltwide Cotton Conference,Annual review of the Texas State Support Committee meeting 2021, and CABANA Food Security Knowledge Exchange Meeting 2022. We havealso published part of our findings in a research article. Thus, results have been disseminated to communities of interest includingthe academic and scientific community working on plant pathology and biology in general, and cotton growers and breeders. What do you plan to do during the next reporting period to accomplish the goals?Our research plan to accomplish goals and objectives will continue according the agency-approved application with slightmodifications. During the next reporting period RNA-seq analysis will continue to identify more RKN and FOV-related resistance genes that can be used to improve cotton. Idenditified candidate resistance geneswill be used to design gene constructs to implement appropriate strategies (e.g., overexpression, inducible expression, RNAi) to assess their potential to confer RKN/FOV resistance to susceptible cotton.

Impacts
What was accomplished under these goals? The potential impact and outcomes from this work will build on the progress to date through conventional breeding to introgress genes for tolerance/resistance to Fusarium oxysporum f.sp. vasinfectum races 1 (FOV1) and 4 (FOV4) and root-knot nematode (RKN), and transcriptomics studies. To date, we have been able to analyze the gene expression profiles of infected cotton entries showing different levels of resistance/susceptibility to both pathogens. Thus, providing a breakthrough understanding of the plant defense mechanisms for FOV and RKN and of the pathogen mechanisms. We identified a series of key genes involved in the resistance mechanisms to RKN which can be used to speed up resistance breeding efforts. Our results are serving as a diagnostic view to understand the genetic and genomic basis for RKN and FOV tolerance/resistance mechanisms in cotton. Building upon efforts of our team to set up the best artificial inoculation protocols and the best time-points for harvesting root tissue for RNA-seq studies at different seedling/plant developmental stages, during this period we worked to generate transcriptome profiles of infected cotton tissues (Objective 2), to conduct comparative RNA-seq dataset analysis, and to identify potential RKN/FOV resistance candidate genes, as presented below. To characterize the cotton transcriptional response to Fusarium wilt disease caused byFOVand the RKNdisease caused byM. incognita,we performed RNA-seq analysis of cotton roots subjected to 6 different treatments: 1) FOV race 1 (FOV1), 2) FOV race 4 (FOV4), 3) RKN, 4) FOV1+RKN, 5) FOV4 +RKN,and 6) non-infected control at 3 and 8 days after infection (dai). To get insights into the molecular mechanisms and the genetic diversity that underlie resistance and susceptibility response to both pathogens, we performed transcriptional profiling of six different cotton entries showing different levels of resistance/susceptibility including 3Gossypium hirsutum(Upland) entriesand 3Gossypium barbadense(Pima) entries. Tested Upland cultivars were comprised by TM-1, Acala NemX (NemX) and PSS-U77B (U77B). Tested Pima cultivars included Pima 3-79 (P3-79), Pima S-6 (PS6) and Pima S-7 (PS7). We first determined differential gene expression in response to the tested infection treatments (RKN, FOV1, FOV4, FOV1+RKN, FOV4+RKN) across allG. hirsutumandG. barbadensegenotypes tested. A gene is considered differentially expressed if its expression changes in all three genotypes tested per species at any of the two times tested (3 or 8 dai) using a change and statistical significance threshold. We detected an important transcriptional response in bothG. hirsutumandG. barbadensecotton entries in response to RKN and RKN/FOV combined treatments. However, data indicated that there is no differential gene expression in response to FOV1 and FOV4 treatments inG. hirsutumentries at any of the two infection times tested, whereas the transcriptional response inG. barbadenseentries under these conditions was very discrete.Interestingly, we detected that a good number of differentially expressed genes was shared between dual infection treatments with the nematode and the fungus (FOV1+RKN and FOV4+RKN) and RKN only. In addition, we detected setsof genes which are specifically activated in response to FOV1+RKN and FOV4+RKN treatments indicating a specific response to FOV in the presence of the nematode. An interesting hypothesis is that RKN treatment might facilitate both FOV1 and FOV4 infections which triggerthe regulation of these additional sets of genes. To test this hypothesis, we carried out Gene Ontology (GO) enrichment of the sets of genes that are regulated in response to RKN, FOV1+RKN and FOV4+RKN treatments to determine the biological processes activated in response to these multiple infection conditions. In the case of RKN treatment, GO analysis indicates that several categories related to the cell cycle and hormonal regulation pathways are activated in bothG. hirsutumandG. barbadensegenotypes tested. This is consistent with previously reported data as nematodes are known to induce cell cycle and manipulate phytohormone pathways for feeding site formation. Other categories related to pathogen infection were enriched as expected including those related to biotic stimulus and hydrogen peroxide. In the case of RKN+FOV1 and RKN+FOV4 we detected the enrichment of several categories including "defense response to fungus", "response to fungus" and "chitin binding" which indicate thatFOV infection was successful and corroborated our hypothesis that RKN facilitates infection of both Fusarium races. These findings arerelevant becauseFOV1 field symptoms on Upland cotton has been associated with co-infestation with RKN in a classic nematode-fungus disease complex, which occurs on the lighter textured sandy soils conducive to RKN problems. On the other hand, although the highly virulent FOV4 strain in California fields hasnot been associated with RKN co-infection, preliminary results from phase 1 of this project showed that there is an additive or synergistic effect on cotton damage between FOV4 and RKN and our RNA-seq analysis confirm theseobservations.We prepared heatmaps for the sets of upregulated genes inG. hirsutumandG. barbadensewhich will aid in the deciphering of gene clusters with similar gene expression behavior among disease-resistant genotypes and time-specific responses. We plan to extend both GO and gene expression analysis to get more insights from the data. To gain more insights on cotton responses specifically to RKN infection, we performed RNA-seqanalysis of threeG. hirsutumcotton entries including SJ-2, WMJJ and NemX that presentlow, intermediate, and high levels of resistance to RKN infestation, respectively. Plants were infected with RKN only and tissue collected at a late stage of the infection. RNA-seq analysis were performed following a traditional pipeline as above.Our results showed thatRKN-resistance is determined by a constitutive state of defense transcriptional behavior that prevails in the roots of the NemX cultivar. NemX responses are enriched in jasmonic and salicylic acid-related genes, two key phytohormones in plant defense responses. These genes included cotton genes coding for disease resistance and receptor proteins linked to RKN-resistance and perception in plants. Interestingly, we identified twoG. hirsutumortholog genes coding for NBS-LRR proteins that have enhanced expression in NemX and mapped to a previously identified RKN-resistance QTL in cotton. We reported these findings in a research article inFrontiers in Plant Science (Ojeda-Rivera et al., 2022, 13;13:858313, doi: 10.3389/fpls.2022.858313). These studies, allowed us to identifya series of key genes involved in the resistance mechanisms to RKN which can be used to speed up resistance breeding efforts. Similar analysis are in process for FOV and preliminary candidate resistance genes have been selected for further biological validation.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Ojeda-Rivera JO, Ulloa M, Roberts PA, Kottapalli P, Wang C, N�jera-Gonz�lez HR, Payton P, Lopez-Arredondo D, Herrera-Estrella L. Root-Knot Nematode Resistance in Gossypium hirsutum Determined by a Constitutive Defense-Response Transcriptional Program Avoiding a Fitness Penalty. Front Plant Sci. 2022 Apr 13;13:858313. doi: 10.3389/fpls.2022.858313. PMID: 35498643; PMCID: PMC9044970. (There was mistake during the editing process and this grant was not included in the list).