Progress 10/01/15 to 09/30/20
Outputs
Target Audience:The target audience for this progress report is plant pathologists, mycologists, crop breeders, and those interested in Alabama agriculture. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training:Throughout this project, laboratory personnel have been trained in techniques used in fungal isolation, culturing, and identification by morphologic and molecular methods. They have gained experience in molecular manipulation of these fungi by gene deletion/complementation/fusion procedures and learned or developed assays to investigate the function of proteins encoded by these genes. Genomic, bioinformatic, and phylogenetic techniques have been developed and utilized in the laboratory. Over the past five years, two graduate students and myself have attended a week-long bioinformatics training session that is held annually at Auburn University. Professional development: As for my professional development, I attended several conferences these past five years that were relevant to the research conducted for this Hatch project. Many of the seminars I attended were pertinent and, in some instances, applicable to the project. The conferences included the 2016 Annual Meeting of the American Phytopathological Society (APS), the 29th and 30th Fungal Genetics Conference at Asilomar, CA, the International Congress of Plant Pathologists 2018 in Boston, the Plant Health 2019, APS Annual Conference in Cleveland, OH, and the 2019 JGI user Meeting in San Francisco, CA. Opportunities for graduate students ofthe laboratory to attend national/interntional conferences were available to enable them to further explore other areas of plant pathology/mycology. Students (number attending in parentheses) attended the 29th Fungal Genetics Conference (1), International Conference of Plant Pathologists (3), the 30th Fungal Genetics Conference (2), the Southern Division of APS Annual Meeting in Gainesville, FL (1), and attended the virtual 2020 Annual APS meeting (1). How have the results been disseminated to communities of interest?In addition to the previously listed publications, results from this research project have been presented and disseminated at several professional meetings to audiences that would be interested in this research. The diversity of the Fov supernumerary chromosomekaryotypes was initially shared at a meeting of plant pathologists that focused on cotton diseases in Tifton, Georgia in June 2016. A presentation detailing the population of Fov in Alabama as well as initial studies on a virulence factor was presented atthe Georgia Association of Plant Pathologists in March 2017. During the 2019 Annual APS meeting in Cleveland, two presentations were made to attendees, one presentation focused on the population structure of Fov in Alabama while the second concerned the CRISPR/Cas9 system developed for Fusarium, where it was estimated that over 150 people attended the seminar. Students in the laboratory were also involved in presenting their data and disseminating the results of this project. A graduate student presented a poster concerning the details ofa virulence factor in Fov at the 29th Fungal Genetics Conference, and two graduate students presented posters at the International Congress of Plant Pathologists 2018 (one presented the Fov genomic data and another presented the gossypol transcriptomic data) while a third student was selected to give an oral presentation regarding the development of the CRISPR/Cas9 ribonucleoprotein gene editing method for Fusarium. The application of the CRISPR/Cas9 system to generate gene fusions (i.e. GFP tagging) was presented in a poster at the 2019 Southern Division APS meeting.At the 30th Fungal Genetics Conference, one student presented a poster concerning the genome analysis of Fov and another student was selected for an oral and poster presentation concerning the identification and characterization of a superoxide dismutase from the fungus that is involved in virulence on cotton. Due to the pandemic this past year, limited opportunities have been available for presenting data, however a student presented data concerning host-specificity for pea in F. vanettenii. In addition, the plasmid containing the Fusarium optimized Cas9 protein has been deposited to the repository Addgene (plasmid ID 112065) for other researchers to request and use. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Research in the laboratory concerning this Hatch Project was primarily focused on the interaction betweenFusarium oxysporumf.sp.vasinfectum(Fov) and cotton and to a lesser extentF. oxysporumf.sp.pisiandF. vanetteniiand garden pea. Impact Statement:A better understanding of the interaction between the fungal pathogen and host plant will enable more efficient disease management strategies, and therefore decreased cost to stakeholders; this project seeks to identify key interactions between the fungus responsible for Fusarium wilt and cotton. 1) Identify the diversity of accessory genomic elements, in particular supernumerary chromosomes, that exist within phytopathogenic fungi. A.Major activities completed/experiments conducted. In an effort to assess the diversity of supernumerary chromosomes ofFovin Alabama, fungi were isolated from cotton plants displaying symptoms indicative of Fusarium wilt over a three-year period. Isolates were subjected to multilocus sequence typing (MLST) to confirm they wereFovand assess the diversity of the pathogens. The diversity of the putative supernumerary chromosomes was assessed by pulse field gel electrophoresis (PFGE).The genome sequence of five different genotypes ofFovisolates was obtained by PacBio sequencing, assembled, and annotated. The linkage between host-specificity and supernumerary chromosomes was first established in isolates ofF. vanettenii. In an effort to further build upon this knowledge, the genomes of 18 additional field isolates were obtained for comparison to the first sequenced isolate. B.Data collected. The MLST data and phylogenetic analysis was conducted for ~180Fovisolates, and PFGE karyotypes of the small chromosomes (<3 Mb) of ~30 isolates were obtained. The genome sequences and annotation of the fiveFovisolates was completed as well as the genome sequence and assembly of 4 of the 18F. vanetteniiisolates has been completed. The remaining 14 are in various stages of sequencing and assembly. C.Summary statistics and discussion of results. Of theFovpopulation in Alabama, MLST data indicated there was a single clade/haplotype responsible for the majority of Fusarium wilt, and there were at least 8 additional haplotypes ofFovisolates from diseased plants. PFGE karyotypes revealed there was high chromosomal diversity, both in size and number, not only between isolates from different haplotypes, but also within a single haplotype. Genome sequencing of the fiveFovisolates revealed the genomes ranged in size from 50.0 Mb to 63.3 Mb and encoded ~18-20,000 putative genes (Seo et al., 2020). There was high variability in the amount of repetitive sequence in the genomes. Comparative genomics revealed all the genomes contained regions that appear to be part of the accessory genome and perhaps supernumerary chromosomes. Further analysis uncovered a ~250 kb syntenic locus that was shared between three of the sequenced isolates and therefore it could be involved in host-specificity on cotton. D.Key outcomes and other accomplishments.The revelation that multiple haplotypes ofFovexist in Alabama, but that there is a single dominant haplotype, indicatesresistance to this haplotype should be prioritized inselective breeding programs. Obtaining genomic data is key to further elucidation of the molecular mechanisms the fungus uses to cause disease on cotton. This data was used as part of objective 2 below. 2) Identify and characterize traits that are conferred by the accessory genome with a specific focus on plant pathogenicity. A.Major activities completed/experiments conducted. Transcriptomic studies were conducted to identify key genes involved in tolerance to gossypol, the phytoalexin (a plantantimicrobial compound synthesized in response to pathogens) produced by cotton. Comparative genomics was used to identify the accessory regions/supernumerary chromosomes in fiveFovgenotypes. PFAM analysis was conducted to identify potential virulence factors. Specific emphasis was placed on small secreted proteins (SSPs), as they could serve as effectors, and secondary metabolite biosynthetic (SMB) clusters. B.Data collected. Transcriptomic data was collected to identify differentially expressed genes in Fov in response to gossypol. The genomic data was analyzed to identify putative SSPsand SMB genes.Fovmutants of genes of interest were generated, and their role in virulence assessed in two different virulence assays on cotton. C.Summary statistics and discussion of results. The transcriptomic data identified 707 genes up regulated for at least one time point, and 68 genes up-regulated at all time points of the study. Importantly, mutants encoding a putativebeta-lactamase and a tannase were reduced in virulence when tested in a whole plant assay. Comparative genome analysis between the fiveFovgenomes and another eleven otherF. oxysporumgenomes identified 79 putative SSPs that were found in at least threeFovgenomes and noF. oxysporumisolates that are not virulent on cotton. Several of these genes were selected to generate mutants, including a cluster of three SSPs that reside beside one another. When this cluster was deleted, the resulting mutant was reduced in virulence on cotton. In addition, a mutant of another putative SSP was reduced in virulence. An extracellular (secreted) superoxide dismutase (FoSod5) was found to be expressed during infection of cotton and deletion of the gene indicated it was important for virulence. This enzyme was able to confer tolerance to ROS compounds and required Cu as a co-factor. Through generation of a GFP fusion, the protein was demonstrated to be secreted, and bound most likely to the cell wall or membrane. In regard to SMB clusters inFov,there were 39to 46SMB clusters in these genomes, and only the race 4 and LA 108 genomes were found to encode SMB clusters in the accessory genome. The numbers of SMB clusters are consistent with otherF. oxysporumgenomes, including those not pathogenic on cotton. D. Key outcomes and other accomplishments. Identification of many putative virulence factors for Fov. 3) Begin to identify external stimulithat facilitate lateral transfer of accessory genomic elements between fungal isolates. A.Major activities completed/experiments conducted. Identification of putative accessory genomic elements through comparative genomics. Developan efficient gene editingsystem to label genes/proteins in the accessory genome scaffolds so they can be monitored for lateral transfer. B.Data collected. Accessory genomic elements were identified in the five sequencedFovgenomes. A CRISPR/Cas9 mediated gene fusion method was developed in the laboratory to enable tagging loci in the accessory genome. C.Summary statistics and discussion of results. The accessory genome of the five sequencedFovisolates ranged in size from 4.11 to14.59 Mb. There was also a considerabledifference in the number of putative genes encoded in the accessory genome ranging from 976 genes to 2702 genes. In order to track transfer of the putative supernumerary chromosomes to evaluate the role of external factors in their later transfer, a CRISPR/Cas9 ribonucleoprotein gene editing method was developed. Proof of concept studies resulting from this researchhave been published (Wang et al., 2018; Wang and Coleman, 2019). Constructs have been made flanking the Cas9 cleavage sitein several scaffolds containing accessory genomic regionswhich willenable insertion of a constitutively active GFP cassette; however due to the end of the project period there have been no labeled supernumerary chromosomes generated. D. Key outcomes and other accomplishments. Development of an efficient gene editing method for Fusarium spp.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Seo S, Pokhrel A, Coleman JJ (2020) The genome sequence of five genotypes of Fusarium oxysporum f. sp. vasinfectum: a resource for studies on Fusarium wilt of cotton. Molecular Plant-Microbe Interactions 33: 138-140.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Wang Q, Pokhrel A, Coleman JJ (prepped) The extracellular superoxide dismutase Sod5 from Fusarium oxysporum is localized in response to external stimuli and contributes to fungal pathogenicity. In revision for Frontiers in Plant Science.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Pokhrel A, Coleman JJ. (2020) Evaluation of host specific virulence factors of Fusarium solani f.sp. pisi for garden pea (Pisum sativum L.). Plant Health 2020, APS Annual Meeting, online.
- Type:
Book Chapters
Status:
Submitted
Year Published:
2020
Citation:
Seo S, Pohkrel A, Wang Q, Coleman JJ. Targeted gene disruption via CRISPR/Cas9 ribonucleoprotein complexes in Fusarium oxysporum. In: Fusarium wilt: Methods and Protocols. Coleman JJ, editor. Methods in Molecular Biology series.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2019
Citation:
Wang, Q (2019) Development of a CRISPR/Cas9 gene editing system for Fusarium oxysporum and characterization of an extracellular superoxide dismutase and its contribition to pathogenicity on cotton. Ph.D. Dissertation, Auburn University.
- Type:
Book Chapters
Status:
Submitted
Year Published:
2020
Citation:
Wang Q, Coleman JJ. CRISPR/Cas9 RNP-mediated gene fusion to assess protein quantification and subcellular localization in Fusarium oxysporum. In: Fusarium wilt: Methods and Protocols. Coleman JJ, editor. Methods in Molecular Biology series
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The target audience for this progress report is plant pathologists, mycologists, crop breeders, and those interested in Alabama agriculture. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this project period laboratory personnel have continued to be trained in techniques used in fungal isolation, culturing, and identification by morphologic and molecular means. They have gained experience in molecular manipulation of these fungi by gene deletion/complementation procedures and learned or developed assays to investigate the function of proteins encoded by these genes primarily focusing on pathogenesis. Additionally, genomic, bioinformatic, and phylogenetic techniques have been developed and utilized in the laboratory. As for professional development, two graduate students and I attended the 30th Fungal Genetics Conference in Pacific Grove. During this meeting we attended several seminars concerning areas that were relevant to this Hatch project. Additionally I attended the JGI User Meeting in San Francisco, CA and the Plant Health 2019, APS Annual Conferencein Cleveland, OH. Topics and presentations at both of these meetings were informative. How have the results been disseminated to communities of interest?In addition to thereported publications, current progress hadbeen reported during the 30th Fungal Genetics Conference. One student presented a poster concerning the genome analysisof Fusarium oxysporum f.sp. vasinfectum and another student was selected for an oral and poster presentation concerning the identification and characterization of a superoxide dismutase from the fungus that is involved in virulence on cotton. In addition, I presented two oral presentations at the Plant Health 2019, APS Annual Conferencein Cleveland, OH, and it is estimated that over 150 attended thepresentation on the use of CRISPR in filamentous fungi. What do you plan to do during the next reporting period to accomplish the goals?This next year we will continue to use comparative genomics to identify regions of the genome that may be of interest. We have generated five mutants that initial assesment indicatesthey are involved in causing disease. We will continue to characterize these potential virulence factors. Additionally, we will try to cure the isolates of putative supernumerary chromosomes to determine if they are involved in host specificity for colonization of agriculturally important crops. Additionally as mentined above, we have begun studies on the accessory genome of F. oxysporum and F. solani that cause disease on pea. In the upcomming year we will begin comparative analyses to identify regions that are shared between these two groups of pathogens.
Impacts
What was accomplished under these goals?
In regards to assessing the role of accessory genomic elements in Fusarium osxysporum f.sp vasinfectum, the causative agent for Fusarium wilt of cotton, we have sequenced and published the data concerning five genomes from isolates of different genotypes. We have begun to identify genes of interest and generate mutants to determine their role in clausing disease on cotton. Initial studies have indicated we have identified several genes that contribute to virulence, and we are begining to further characterize them. Additionally, this past year we have begun a new project to assess the accessory genome of F. oxysporum and F. solani isolates that are virulent on garden pea. Comparative genomics will be utilized to further determine unique regions that may contribute to virulence.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Wang Q, Coleman JJ (2019) CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum. Fungal Genetics and Biology 126: 17-24.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Wang Q, Coleman JJ (2019) Progress and challenges: development and implementation of CRISPR/Cas9 technology in filamentous fungi. Computational and Structural Biotechnology Journal 17: 761-769.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2020
Citation:
Seo S, Pokhrel A, Coleman JJ (in press) The genome sequence of five genotypes of Fusarium oxysporum f. sp. vasinfectum: a resource for studies on Fusarium wilt of cotton. Molecular Plant-Microbe Interactions
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Coleman, JJ. CRISPR/Cas9 gene editing in Fusarium oxysporum. Plant Health 2019, APS Annual Conference, Cleveland, OH.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Coleman, JJ. Fusarium wilt of cotton in Alabama. Plant Health 2019, APS Annual Conference, Cleveland, OH.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Seo, S. Pokhrel, A. and Coleman, JJ. Comparative genomic analysis of Fusarium wilt isolates associated with virulence on cotton. 30th Fungal Genetics Conference, Pacific Grove, CA.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Wang, Q. and Coleman, JJ. Sod5 from Fusarium oxysporum f. sp. vasinfectum contributes to fungal pathogenicity and localizes based on environmental stimuli. 30th Fungal Genetics Conference, Pacific Grove, CA.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The target audience for this progress report is plant pathologists, mycologists, and those interested in Alabama agriculture. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this project period laboratory personnel have continued to betrained in techniques used in fungal isolation, culturing, and identification by morphologic and molecular means. They have gained experience in molecular manipulation of these fungi by gene deletion/complementation procedures and learned or developed assays to investigate the function of proteins encoded by these genes primarily focusing on pathogenesis. Additionally, genomic, bioinformatic, and phylogenetic techniques have been developed and utilized in the laboratory. As for professional development, two graduate students and I attended the Interantional Congress of Plant Pathologists 2018. During this meeting we attended several seminars concerning areas that were relevant to this Hatch project. How have the results been disseminated to communities of interest?In addition to the previously reported publication, current progress has been reported during the International Congress of Plant Pathologists 2018. One student presented a poster concerning the response ofFusarium oxysporum f.sp. vasinfectum when challenged with the cotton phytoalexin gossypol and another student was selected for an oral presentation based on the development of the CRISPR/Cas9 ribonucleoprotein gene editing system. In addition, the plasmid containing the Fusarium optimized Cas9 protein has been deposited to the repositoryAddgene (plasmid ID 112065) for other researchersto request and use. What do you plan to do during the next reporting period to accomplish the goals?This next year we will continue to use comparative genomics to identify regions of the genome that may be of interest. Genes within these regions will be mutated and determined if they play a role in virulence. Additionally, we will try to cure the isolates of putative supernumerary chromosomes to determine if they are involved in host specificity for colonization of agriculturally important crops.
Impacts
What was accomplished under these goals?
The interaction between a pathogen and host plant are constantly evolving. One mechanism by which fungal pathogens can quickly alter their interactions is through the presence of extra chromosomes which carry host-specific virulence traits. This project aims to identify and assess the diversity of these extra chromosomes in the genus Fusarium, a group of plant pathogens with a broad host range. Through comparative genomics the laboratory has identified several loci that reside in only in the genomes of F. oxysporum isolates able to cause disease on cotton. Some of these lociencompass~20 putative genes arranged syntenically suggesting that have a similar origin and are currently the focus of functional studies. To aid in functional analysis we have optimized a CRISPR/Cas9 medicted transformation system. Additionally, we have begun functional analysis of genes that are the highest upregulated in the presence of gossypol, the phytoalexin produced by cotton in response to pathogens.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Wang Q, Cobine PA, Coleman JJ (2018) Efficient genome editing in Fusarium oxysporum based on CRISPR/Cas9 ribonucleoprotein complexes. Fungal Genetics and Biology 117: 21-29.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Pokhrel A. (2018) Transcriptomic analysis to identify candidate genes conferring gossypol tolerance in Fusarium oxusporum f. sp. vasinfectum. M.S. Thesis, Auburn University.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Coleman J, Pokhrel A (2018) Transcriptomic analysis for differentially expressed genes in response to the phytoalexin gossypol in Fusarium oxysporum f.sp. vasinfectum. Phytopathology 108, S1.223.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Wang Q, Cobine P, Coleman JJ (2018) Efficient genome editing in Fusarium oxysporum beased on CRISPR/Cas9 ribonucleoprotein (RNP) complexes. Phytopathology 108, S1,247.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The target audience for this progress report is plant pathologists, mycologists, and those interested in Alabama agriculture. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this project period laboratory personnel have been trained in techniques used in fungal isolation, culturing, and identification by morphologic and molecular means. They have gained experience in molecular manipulation of these fungi by gene deletion/complementation procedures and learned or developed assays to investigate the function of proteins encoded by these genes primarily focusing on pathogenesis. Additionally, genomic, bioinformatic, and phylogenetic techniques have been developed and utilized in the laboratory. As for professional development, a graduate student and I attended the 29th Fungal Genetics Conference in March. During this meeting weattended several seminars concerning areas that were relevant to this Hatch project. How have the results been disseminated to communities of interest?A seminar detailing some of the datawas presented to plant pathologists at the Georgia Association of Plant Pathologistsin March 2017. Additionally, a graduate student presented a posterat the 29th Fungal Genetics Conference concerning some of the data generated as part of this Hatch project. What do you plan to do during the next reporting period to accomplish the goals?This next year we will continue to use comparative genomics to identify regions of the genome that may be of interest. Genes within these regions will be mutated and determined if they play a role in virulence. Additionally, we will try to cure the isolates of putative supernumerary chromosomes to determine if they are involved in host specificity for cotton.
Impacts
What was accomplished under these goals?
The interaction between a pathogen and host plant are constantly evolving. One mechanism by which fungal pathogens can quickly alter their interactions is through the presence of extra chromosomes which carry host-specific virulence traits. This project aims to identify and assess the diversity of these extra chromosomes in the genus Fusarium, a group of plant pathogens with a broad host range. This past year the laboratory has generated and assembled the genome sequences of five of the F. oxysporum isolates. Comparative analysis is currently underway to identify regions that might be supernumerary chromosomes. These regions may play a role in causing disease on cotton. Additionally, we have determined the transcriptional response of one of these isolates in response to treatment with the cotton phytoalexin gossypol. As we have begun to characterize some of these genes of interest, the laboratory has developed a CRISPR/Cas9 based gene editing system for members of the Fusarium genus.
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:The target audience for this progress report is plant pathologists, mycologists, and those interested in Alabama agriculture. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this project period laboratory personnel have been trained in techniques used in fungal isolation, culturing, and identification by morphologic and molecular means. They have gained experience in molecular manipulation of these fungi by gene deletion/complementation procedures and learned or developed assays to investigate the function of proteins encoded by these genes primarily focusing on pathogenesis. Additionally, genomic, bioinformatic, and phylogenetic techniques have been developed in the laboratory. As for professional development, I attended the 2016 Annual Meeting of the American Phytopathological Society in August. During this meeting I attended several seminars concerning areas that were relevant to this Hatch project. How have the results been disseminated to communities of interest?A seminar detailing some of the data on supernumerary chromosomes was presentedto plant pathologists that work on cotton diseases in Tifton, Georgia in June 2016. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The interaction between a pathogen and host plant are constantly evolving. One mechanism by which fungal pathogens can quickly alter their interactions is through the presence of extra chromosomes which carry host-specific virulence traits. This project aims to identify and assess the diversity of these extra chromosomes in the genus Fusarium, a group of plant pathogens with a broad host range. This past year the laboratory has isolated members of the F. oxysporum species complex and used phylogenetic analysis to determine how these isolates are related to each other. The chromosomal profiles of these isolates were analyzed by pulsed-field gel electrophoresis revealing a high degree of variability in the number and size of small chromosomes, which may represent some of the extra chromosomes conferring pathogenicity. To further determine the role these chromosomes in plant pathogenicity, we have selected fives isolates for genome sequencing and will begin to determine their function by comparative genomics. Currently, the extracted DNA is in the process of being sequenced. Once genes of interest are identified from the genomic sequence, the laboratory will eventually generate mutants which lack these genes. In preparation for these experiments, the laboratory has developed a new transformation system for use with Fusarium which is currently being further optimized to facilitate generation of mutants.
Publications
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