Location: Molecular Plant Pathology Laboratory
2021 Annual Report
Objectives
Identify, sequence, and characterize plant and pest genes associated with resistance of sugar beet to the root maggot and important pathogens using transcriptomic databases prepared from interactions of this pest with its resistant and susceptible hosts, and use the discovered genes to enhance plant disease and pest tolerance traits. [NP301, C1, PS1A, PS1B]
Profiling of sugar beet genes in a resistant and susceptible host following infestation by the sugar beet root maggot yielded gene libraries enriched for genes that are modulated by the host-pest interaction. To better understand the role of sugar beet genes in resistance, identified genes need to be functionally characterized. Expression of the genes in a heterologous and a model sugar beet root system is an approach for determining their function in disease and pest resistance. Promoters of the identified resistance genes provide a pool of temporal and tissue-specific promoters that have the potential to preferentially target beneficial gene expression to sites attacked by pests and pathogens and to rapidly block disease onset and progression. Complementary studies with root maggot genes will be valuable tools for genetic manipulation of the host plant to specifically target essential pest genes needed for effective interaction with the host. Identified sugar beet and pest genes will provide new tools for designing effective, environmentally sound, biotechnologically based strategies to improve insect and disease resistance in sugar beet and other important crop plants. In addition to genetic modification and genome editing technologies, genes can be used as genetic markers in conventional breeding programs to select sugar beet germplasm with improved resistance and to screen germplasm for complementary insect disabling traits to develop safe control measures.
Design, validate, and implement genome editing approaches for sugar beet to improve disease and pest resistance in commercially important genotypes. [NP301, C3, PS3A]
Genome editing technology (CRISPR/Cas9) is anticipated to provide enhancements in crop yield, shelf life, nutritional content, physical appeal, production of specialty chemicals, and abiotic and biotic stress tolerance. Plant scientists have quickly adopted genome editing technology to gain insights into plant biological processes and for altering plant traits. Mutagenesis, gene knockout and gene knock-in are all easily and rapidly achievable with this technology. Many of the technical challenges associated with currently used plant transformation technology, such as position effects and copy number variability, can be alleviated or minimized with genome editing approaches. Genome editing of sugar beet has not been reported. We will design and validate genome editing vectors using sugar beet resistance genes characterized in Objective 1 above to elucidate their role in resistance mechanisms. This newly generated knowledge will lead to genome editing strategies for improving disease and pest resistance in elite, commercially important sugar beet and other crop plants.
Approach
Sugar beet root defense genes incited by the root maggot, a destructive pest of sugar beet, will be functionally characterized in sugar beet and Nicotiana using molecular transformation approaches and genome editing (CRISPR-Cas9). Understanding of the genes’ role in defense will be used to develop screening protocols of sugar beet germplasm for resistance traits and to devise novel strategies for pest and disease control. The role of two genes that were demonstrated to enhance resistance and that preferentially respond to root maggot feeding in a resistant germplasm will be evaluated for resistance to insects and phytopathogens. Genetically modified sugar beet roots and Nicotiana plants that were demonstrated to be resistant to several different insects will be bioassayed for resistance to sugar beet fungal pathogens, and conversely modified plants that were shown to be resistant to several phytopathogens will be screened for resistance to insect pests. One of the sugar beet genes that codes for a serine proteinase inhibitor (PI; BvSTI) was shown to enhance resistance to several insect pests (beet and fall armyworm, tobacco hornworm). Another sugar beet gene that codes for a cell wall polygalacturonase inhibitor (PGIP, BvPGIP) was shown to enhance fungal resistance to Fusarium solani, Rhizoctonia solani and Botrytis cinerea. BvSTI is a wound inducible serine PI with specificity for the root maggot digestive enzymes that mediate release of nutrients from ingested plant tissues. BvPGIP codes for a leucine-rich repeat glycoprotein PGIP that is associated with cell wall structure and plant defense responses. A group of sugar beet genes encoding enzymes for fatty acid (lipid) biosynthesis were also isolated using a transcriptomic approach and shown to increase lipid accumulation by up to 45% in sugar beet roots and Nicotiana plants. To evaluate the effect of elevated lipids on resistance, plants producing the recombinant fatty acid transcription factors will be bioassayed for insect and fungal resistance using similar approaches as described above. To more precisely target the expression of beneficial genes to root cells and tissues most prone to pest and pathogen attack, BvPGIP and BvSTI gene promoters will be characterized in sugar beet hairy roots and model plants. Expression of a GUS reporter gene fused to the sugar beet promoters will be evaluated in response to various biotic and abiotic stresses that include insect infestation, phytopathogen infection and mechanical wounding. In complementary studies of insect responses, root maggot genes that were shown to be important for interaction of the pest with resistant or susceptible sugar beet roots will be characterized. Profiled, sequenced and functionally annotated root maggot genes will provide new knowledge of how insects adapt to host plants and surmount host resistance. With the newly discovered knowledge of sugar beet resistance and root maggot genes, genome editing approaches will be designed to improve plant resistance. Identified pest and root these genes will also be used to screen elite sugar beet germplasm for inherent resistance traits.
Progress Report
The lab is currently being set up from scratch after the retirement of Dr. Ann Smigocki since the computers, equipment, and biologicals related to the project no longer exist. The setting up of the lab includes various work orders and ordering of equipment and supplies based on the needs of the project. Regarding obtaining materials (sugar beet seeds and samples), I have reached out to Dr. Linda Hanson USDA-ARS, Dr. Barbara Hellier USDA-ARS, Dr. Chenngen Chu USDA-ARS, Dr. Larry Campbell (USDA-ARS [retired]), and others in the sugar beet community for seeds for the sugar beet hairy root transformation and inquired about the availability of pathogen stocks. Furthermore, to bulk up seeds, we have communicated with West Coast Beet Seed Co., who performs the task. Those communications are ongoing. The collaboration with Dr. Chenggen Chu is in relation to the sugar beet root maggot. In collaboration, he is pulling larvae out of their storage in the refrigerator and has put them in growth chambers to induce the pupation to initiate experiments that will lead to the infection of the roots of susceptible and resistant genotypes for RNA sequencing experiments to identify candidate resistance genes.
In addition to these efforts, promoter sequences for a serine proteinase inhibitor (BvSTI) and cell wall polygalacturonase inhibitor (BvPGIP) are being identified by bioinformatics. The analyses, in part, used the double haploid genotype KWS2320 sugar beet genome and the newer five-generation inbred 'C869' (PI 628755) genome released as ‘EL10’ (PI 689015). To facilitate promoter analyses, a literature research has been performed to identify the best cloning vector for plant gene expression of the beta galacturonidase (GUS) reporter, including pMD162, pMD163, pMD164. Furthermore, sugar beet gene accessions have been used to identify the BvSTI and BvPGIP sequences for gene cloning and transgenic research in sugar beet hairy roots involving the aforementioned pRAP15 and pRAP17 plasmids. Through bioinformatics analyses, the genes have been identified. For the Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/Cas9 experiments, plasmids to be used in the experiments have been identified through Addgene, including p201N: Cas9 (Addgene plasmid 59175), NptII selectable marker, p201H: Cas9 (Addgene plasmid 59176), hygromycin phosphotransferase, p201B:Cas9 (Addgene plasmid 59177), bar-phosphinothricin resistance and p201G:Cas9 (Addgene plasmid 59178), (eGFP) reporter. The guide RNA would be engineered using the pUC gRNA shuttle plasmid obtained from Addgene (plasmid 47024). The gRNA can be added directly into the CRISPR plasmid, so both the CRISPR and gRNA are on the same plasmid to facilitate gene editing. Inquiries have also been made to Creative Biogene to have CRISPR/Cas9 gene editing done of the 3 polygalacturonase inhibitor (PGIP) Nicotiana benthamiana genes as a proof of concept to show that the gene knockouts would increase their susceptibility to the sugar beet root maggot. DNA sequences for plasmids to be used in the planned experiments have been submitted to the Genbank archive. The submitted sequences are PRJNA734620 and PRJNA734623.
Accomplishments
