Location: Molecular Plant Pathology Laboratory
2022 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
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 (CRISPRCas9).
Since the molecular transformation approaches require development, efforts have been made to identify and obtain source material. Regarding obtaining sugar beet seeds and samples, I have reached out to the sugar beet community for seed stocks. I have been able to obtain some seed of sugar beet seed stocks including C869 (PI 628755) and EL10 (PI 689015) which require bulking up. Additional studies have been started in collaboration with a USDA-ARS collaborator to screen 2,000 sugar beet lines for resistance to sugar beet root maggot. He is retrieving larvae out of their storage in the refrigerator and has put them in growth chambers to induce 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 the screen, we am involved in experiments whereby RNA will be isolated from susceptible and resistant roots and used for RNA sequencing to identify additional candidate defense genes.
Accomplishments
1. Root cell expressed gene function in plant defense. The plant secretion apparatus transports proteins to the required location for pathogen defense. The conserved oligomeric Golgi (COG) complex, composed of 8 protein-encoding genes is important in this function. Research identified how the expression of these genes is regulated. To understand the gene regulation better, gene expression studies have identified genes that function in circadian gene regulation that function in defense in the root to a pathogen. To further understand gene regulation better, genes functioning in microbe perception and signal transduction were studied and demonstrated to have a defense role to a root pathogen. Experiments show that these genes can be transferred to other crop species to generate a defense response to other root pathogens. The work has resulted in as great as a 97% decrease in root infection. The impact/outcome is the advance in knowledge demonstrating of how circadian fluxes in gene expression and other processes alter defense gene expression and cell wall composition during defense in the root that previously did not exist.
Review Publications
Khatri, R., Pant, S.R., Sharma, K., Niraula, P.M., Lawaju, B.R., Lawrence, K.S., Alkharouf, N.W., Klink, V.P. 2022. Glycine max homologs of doesn't make infections 1, 2, and 3 function to impair heterodera glycines parasitism while also regulating mitogen activated protein kinase expression. Frontiers in Plant Science. 13:842597. https://doi.org/10.3389/fpls.2022.842597.
Klink, V.P., Lawaju, B.R., Sharma, K., Niraula, P.M., Alkharouf, N.W., Lawrence, K.S. 2022. The conserved oligomeric Golgi (COG) complex functionality in relation to plant defense. Journal of Plant Interactions. 17:344-360. https://doi.org/10.1080/17429145.2022.2041743.
Niraula, P.M., Mcneece, B.T., Sharma, K., Alkharouf, N.W., Lawrence, K.S., Klink, V.P. 2022. The central circadian regulator CCA1 functions in Glycine max during defense to a root pathogen, regulating the expression of genes acting in effector triggered immunity (ETI) and cell wall metabolites. Plant Physiology and Biochemistry. 185:198-220. https://doi.org/10.1016/j.plaphy.2022.05.028.
Klink, V.P., Alkharouf, N.W., Lawrence, K.S., Lawaju, B., Sharma, K., Niraula, P., Mcneece, B.T. 2022. The heterologous expression of conserved Glycine max (soybean) mitogen activated protein kinase 3 (MAPK3) paralogs suppresses Meloidogyne incognita parasitism in Gossypium hirsutum (upland cotton). Transgenic Research. https://doi.org/10.1007/s11248-022-00312-y.
