Performing Department
(N/A)
Non Technical Summary
Soybean cyst nematodes (SCN) pose a substantial threat to soybean production in the US, resulting in annual yield losses exceeding $1.5 billion. The prolonged reliance on the PI88788 (rhg1) resistance source over the past three decades has caused "race shift" in SCN populations, leading to more virulent races. Therefore, it is imperative to identify and develop additional SCN resistance sources independent of rhg1 and Rhg4 to combat this ever-changing pest. Through our prior USDA-NIFA supported research, we identified qSCN10 resistant QTL that harbors GmbZIP10 gene. Remarkably, significant allelic variations in GmbZIP10 strongly correlated with differential gene expression in response to SCN infection. Notably, the increased expression of GmbZIP10 in soybean composite transgenic hairy roots demonstrated enhanced SCN resistance compared to the susceptible allele, offering a promising strategy for engineering robust and broad-based SCN resistance that operates independently of rhg1/Rhg4. Hence, our integrated genomics and gene-editing research aims to enhance broad-based SCN resistance.The outcomes of our research hold the promise of not only enhancing our understanding of the molecular basis of SCN resistance but also paving the way for unconventional and sustainable strategies to combat this formidable and ever-evolving soybean pest in the United States.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Goals / Objectives
Soybean cyst nematodes (SCN) pose a substantial threat to soybean production in the US, resulting in annual yield losses exceeding $1.5 billion. The prolonged reliance on the PI88788 (rhg1) resistance source over the past three decades has caused "race shift" in SCN populations, leading to more virulent races. Therefore, it is imperative to identify and develop additional SCN resistance sources independent of rhg1 and Rhg4 to combat this ever-changing pest. Through our prior USDA-NIFA supported research, we identified qSCN10 resistant QTL that harbors GmbZIP10 gene. Remarkably, significant allelic variations in GmbZIP10 strongly correlated with differential gene expression in response to SCN infection. Notably, the increased expression of GmbZIP10 in soybean composite transgenic hairy roots demonstrated enhanced SCN resistance compared to the susceptible allele, offering a promising strategy for engineering robust and broad-based SCN resistance that operates independently of rhg1/Rhg4. Hence, our integrated genomics and gene-editing research aims to enhance broad-based SCN resistance. Our objectives encompass: (1) Functional validation of a novel GmbZIP10 gene and cis-regulatory elements through CRISPR knock-outs, (2) Engineering SCN resistance through native and constitutive promoters in susceptible genotypes, (3) Deciphering transcriptional regulation of GmbZIP10, (4) Identification of genetic circuits and genetic mechanism of SCN resistance using single-cell transcriptomics studies. The outcomes of our research hold the promise of not only enhancing our understanding of the molecular basis of SCN resistance but also paving the way for unconventional and sustainable strategies to combat this formidable and ever-evolving soybean pest in the United States.?
Project Methods
Obj 1: Functional validation of a novel GmbZIP10 gene and cis-regulatory elements through CRISPR knock-out.a) Functional validation of GmbZIP through CRISPR knockout: To functionally validate the role of GmbZIP10, we will deploy a strategy to knock out GmbZIP10 gene by using CRISPR/Cas9 with dual guide RNA approach in resistant lines PI 567516C and MgNIL and susceptible line Magellan. Briefly, we will design two guide RNAs in the exon of GmbZIP10 gene by using CRISPR-P tool (Lei et al., 2014) . Codon optimized AtCas9 gene will be expressed under the control of GmUbi promoter, while gRNAs will be expressed under constitutive CmYLCV promoter. The final binary vector harboring AtCas9, gRNAs and marker gene cassettes will be transformed into Agrobacterium rhizogenes strain K599. Later transgenic composite roots will be developed in all soybean genotype as per protocol (Fan et al., 2020). Following the robust root biomass (approx. 3 weeks after transformation), roots expressing reporter gene will be kept intact on plant while non transgenic roots will be removed to increase the chance of transgenesis and potentially gene knockout. Subsequently these roots will be infected with the SCN races to assess SCN resistance in soybeanand as detailed in Patil et al., (2019).b) Functional validation of cis regulatory elements in susceptible genotypes: Through haplotype analysis, we identified differential alleles in susceptible and resistant genotypes including two large deletions of 28 bp and 51 bp in the promoter region of GmbZIP10 is a resistant line PI 567516C. Therefore, to determine whether those deletions have role in SCN resistance, we propose to modify promoter of GmbZIP10 in susceptible W82/Magellan by eliminating those cis regulatory regions by using CRISPR/Cas9 technology. We will employ a dual gRNA approach to precisely eliminate each deletion separately or in combination. We will design two gRNAs flanking each cis regulatory region, followed by cloning those gRNAs under CmYLCV promoter. Whereas AtCas9 will be cloned under the control of GmUbi promoter. All the cassettes will be assembled in a final binary vector and subsequently transformed into A. Rhizogenes strain K599. We will assess SCN phenotype by developing transgenic composite roots in W82/Magellan.Obj. 2: Engineering SCN resistance through native and constitutive promoters in susceptible genotypes. a) Expressing GmbZIP10 from PI 567516C (R) using its native promoter: The GmbZIP10 promoter from PI 567516 harbors several allelic variations in cis regulatory regions. These SNPs and deletions may be contributing to higher expression of GmbZIP10 during SCN infection. Using these constructs, we expect to impart resistance phenotype. The entire 2.5 kb promoter of GmbZIP10 from PI 567516C (SCN resistant) will be used to express GmbIZP10 gene in W82/Magellan hairy roots followed by SCN infection and phenotyping.b) Expressing GmbZIP10 from Magellan (S) using PI 567516C promoter: By utilizing the PI 567516C promoter to drive the expression of GmbZIP10 from Magellan, we can gain a better understanding of whether the resistance of PI 567516C to SCN is attributed to the two deletions in the promoter, or SNPs in GmbZIP10 CDS, or a combination of both factors.c) Expressing GmbZIP10 from Magellan (S) using its own promoter: In this experiment, we will utilize Magellan's native promoter and GmbZIP10 sequence to express in Magellan/W82, allowing us to confirm its role in SCN susceptibility. This experiment will serve as internal control, representing SCN resistance that remains unchanged.d) Overexpression of PI 567516C GmbZIP10 in Magellan under strong constitutive promoter: Having identified that GmbZIP10 in PI 567516C and NILs imparts moderate to complete resistance against multiple SCN races, a question arises: Can achieving a 2 to 10-fold increase in GmbZIP10 expression lead to complete resistance against multiple races? To explore this, we will conduct an overexpression experiment by cloning GmbZIP10 from PI 567516C under robust and constitutive GmUbi promoter. This construct will be used to develop transgenic hairy roots W82/Magellan background followed by SCN phenotyping.Obj. 3: Deciphering transcriptional regulation of GmbZIP10To study the mechanism and gene regulatory networks through which it interacts with other genes and regulates SCN resistance, we will employ Chromatin immunoprecipitation- sequencing (ChIP-SEQ) (Kaufmann et al., 2010).To determine target genes of GmbZIP10, we will use GmUbi:Bzip10-GFP construct. Moreover, only GFP expressed under the control of GmUbi will serve as control (GmUbi:GFP). Subsequently constructs will be transformed to A. rhizogenes and later we will generate transgenic composite roots as described by (Fan et al., 2020). ChIP-SEQ will be performed on composite hairy roots with SCN infection (48 hr. and 5 days after SCN infection) and without SCN infection (control). ChIP assay will be performed as per protocol by Kaufmann et al., (2010).We will correlate ChIP-seq data with single cell RNA seq data to illustrate direct and indirect target genes regulated by GmbZIP10 TF during SCN infection. Based on the anticipated role of identified target genes of GmbZIP10 TF, those target genes will be further characterized through systematic overexpression and CRISPR based gene knockout (elaborated in Obj, 1 and 2) in soybean hairy roots. SCN resistance will be measured by including positive, negative and test constructs. Each experiment will be performed in biological and technical replications.Obj. 4: Identification of genetic circuits and genetic mechanism of SCN resistance using single-cell transcriptomics studies.To decipher the gene-transcriptional regulatory network during SCN infection and progression of cyst structures, SCN resistant (MgNILs and PI567516C) and susceptible (Magellan and/or W82) lines will be used. Single-cell trajectories analysis will be studied during SCN infection via integrating Monocle3 and Slingshot toolkits. Differentially expressed genes (DEGs) will be utilized in the enrichment analysis using GProfiler to understand the biological processes, molecular functions, and cellular components. In addition, the KEGG and STRINGdb databases will be used to perform network analyses. We will conduct gene regulatory network (GRN) analysis using the gene regulatory graph neural network (GRGNN) methodology. GRGNN will provide an end-to-end gene regulatory network utilizing the publicly available RNA-Seq datasets as well as those generated in this project. We will also apply GENIE3 toolkit for gene regulatory analysis to prioritize the differentially expressed genes and transcription factors. Finally, genes identified from this analysis will be validated using gene knockout (CRISPR tools) and over-expression studies and correlated with GmbZIP functional analysis (detailed in Obj 1 and 2).