Location: Molecular Plant Pathology Laboratory
2024 Annual Report
Objectives
Objective 1. Devise and develop advanced biotechnological approaches to address and improve SB pest and disease resistance, abiotic factors, yield, and quality.
Objective 2. Decipher and understand molecular mechanisms of Sugarbeet root defense responses to pest and pathogen attack.
Sub-objective 2A. Perform gene expression in SB to identify genes that relate to SB disease response.
Sub-objective 2B. Perform gene expression studies in related systems that relate to the SB systems.
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
In support of Objective 2, larvae of the sugar beet root maggot (SBRM), Tetanops myopaeformis, were sent for DNA isolation and subsequent chemical genome sequencing. SBRM genome sequence knowledge is important due to the limited natural resistance of sugar beet (SB), B. vulgaris, to its pathogens, including the SBRM. This problem necessitates an understanding of the genome to facilitate generating knowledge of its basic biology, including the interaction between the pathogen and its host(s). After the chemical sequencing of the genome, analyses were done that led to the de novo assembled draft genome sequence of T. myopaeformis, isolated from field-grown B. vulgaris from North Dakota. The SBRM genome sequence TmSBRM_v1.0 will be valuable for molecular genetic marker development to facilitate host resistance gene identification and knowledge. The genome would allow for the identification of insect effectors such as polygalacturonases (PGs) that both degrade cell walls and impair defense responses. The genome would aid in the understanding of SB polygalacturonase inhibiting protein (PGIP) as they relate to successful defense processes, and development of new control strategies for this pathogen, the relationship to model genetic organisms like Drosophila melanogaster while aiding in agronomic improvement of sugar beet for stakeholders while also providing information on the relationship between the SBRM and climate change. The DNA sequencing project resulted in covering the genome 94 times, with a total of 6,356,906 raw reads; a total read length of 71,844,227,661 bp; 11,313 8,294 N50/N90 reads, respectively; and a 40.2% GC richness. Furthermore, regarding the assembly statistics, the genome is 414,327,873 bp, produced through 8,228 contigs having57,402 contig N50 with the largest contig being 573,329 bp yielding a mean coverage of 94x. This knowledge is foundational and transformative for the benefit of agriculture in relation to the control of SBRM. The data is available freely and publicly. The information provides the basis for molecular and genetic control of the SBRM which did not exist prior to the availability of its genome. The data is available for the annotation of genome elements of the SBRM. With the assembled data in hand, we were then in a position to determine the genes and other elements that compose the genome through its annotation. The annotation of the T. myopaeformis, SBRM genome, TmSBRM_v1.0, provides data that researchers can use to understand the SBRM’s biology. The information allows for knowledge on chromosome structure, genome organization, maintenance, regulation, and the understanding of gene expression including sex specific gene expression regulation. Furthermore, the annotation is useful for understanding insect evolution, as well as the development and evolution of a pathological niche, and aids in the understanding of host selection, ecology, and climate change. The SBRM genome annotation permits the identification of essential pathogen genes whose function can be impaired through CRISPR/Cas9 gene editing and other means, resulting in its control which would benefit stakeholders. The annotation of the TmSBRM_v1.0 reference genome allows for a better scientific investigation of the insect, and related insect species that have pathogenic life cycles. The analysis identified 28,276 genes in the SBRM genome. There were 21,206 with a Blast hit; 7,070 with no blast hit; there were 890 having a Blast hit (uncharacterized+ hypothetical) and 20316 Blast hit (characterized genes. The number of species matches to the SBRM proteins were 351 different organisms with the most highly matched proteome being to the genetic model insect, Drosophila melanogaster (n = 6,697 different proteins). The annotated genome will allow scientists to benefit stakeholders through the use of the sequenced genome to identify genes that can be inactivated by targeted mutagenesis, RNAi or chemicals that eliminate the SBRM as a pathogen of sugar beet. Furthermore, genes could be activated or introduced that could control the SBRM. The research could also lead to the production of targeted trapping methodologies to control the SBRM. Management strategies designed to better understand the life cycle of the SBRM for better control or on-site molecular analysis are envisioned to be produced through the availability of the SBRM genome and its annotation.
Accomplishments
1. Determined the genome sequence of the sugar beet root maggot (SBRM), Tetanops myopaeformis. The sugar beet root maggot (SBRM), Tetanops myopaeformis, is a devastating insect pathogen of sugar beet, Beta vulgaris, ssp vulgaris (B. vulgaris). Sugar beet is an important food crop, being one of only two plants globally from which sugar is widely produced, and accounting for 35% of global raw sugar with an annual farm value of $3 billion in the United States alone. Determination of the SBRM genome provides critical knowledge on how to control the SBRM, a big priority for sugar beet growers. Control of this pathogen for agricultural benefit would be obtained by having a sequenced genome which prior to this project did not exist.
2. Annotated the sequenced genome of the sugar beet root maggot (SBRM), Tetanops myopaeformis. The annotation of the Tetanops myopaeformis, sugar beet root maggot (SBRM) genome, TmSBRM_v1.0, provides data that researchers can use to understand the SBRM’s biology. The information allows for gaining knowledge on chromosome structure, genome organization, maintenance, and regulation. The information allows for an understanding of gene expression including sex specific gene expression regulation. Furthermore, the annotation is useful for understanding insect evolution, as well as the development and evolution of a pathological niche. The information also aids in the understanding of host selection, ecology, and climate change. The SBRM genome annotation aids in other aspects of improving the understanding of their biology, and agronomic impact(s).
Review Publications
Alkharouf, N., Chu, C.N., Klink, V.P. 2024. An assembly of genomic sequences of the sugar beet root maggot Tetanops myopaeformis, TpSBRM_v1.0. Data in Brief. Article e110298. https://doi.org/10.1016/j.dib.2024.110298.
Acharya, S., Alkharouf, N., Chu, C.N., Klink, V.P. 2024. The annotation of genomic dataset sequences of the sugar beet root maggot Tetanops myopaeformis, TmSBRM_v1.0. Data in Brief. Article e110710. https://doi.org/10.1016/j.dib.2024.110710.