Location: Molecular Plant Pathology Laboratory
2020 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
For Objectives 1 and 2, in FY2019, studies were initiated to determine if transgenic plants and sugar beet hairy roots that express the sugar beet BvPGIP1 and BvPGIP2 genes will be more resistant to fungal pathogens and insect pests and to design genome editing vectors for sugar beet resistance genes. In collaboration with scientists at Michigan State University, a range of Rhizoctonia solani isolates were screened on three lines of Nicotiana benthamiana transformed with the BvPGIP genes. The resulting screen revealed differential responses between transformed and untransformed plant lines with the diverse isolates. In addition, one of the isolates was more aggressive on N. benthamiana than reported on dry bean and sugar beet. Additional screening will be conducted in the future. Preliminary microscopic observations on disease development in the root suggest that infection is not evenly distributed across the root. A patent was issued in FY2019 for work resulting from cooperative research with an industry partner for the "Development of Genetically Altered Plants Producing Fatty Acids". The plants generated displayed increased resistance to various insects. Patent No: US 10,415,051 B2.
Accomplishments
