Project Proposal - PMB

PHD WALSH FELLOWSHIP

ResBarley: Enhancing biotic stress resilience in barley

PHD PROJECT Proposal

Ukoji Stepheine Onyinyechi 25th July 2024

OUTLINE

Introduction

Work Package 1

Work Package 2

Work Package 3

Project work flow

This research project aims to

Specific

General

• Identify up to 10 bona fide S-genes in barley/leaf scald pathosystem
• Generate barley lines with dysfunctional S-genes.
• Evaluate the resilience of the engineered barley lines to leaf scald and other relevant pathogens.
• Evaluate the agronomic performance and malting properties of the new lines to explore any tradeoffs of disrupting the selected S-genes

Utilize genes previously identified to be associated with to leaf scald disease in barley to develop resistant barley lines using NGT.

introduction

Figure 1: Area of some selected cereals and potatoes cultivation in Ireland

Relevance of barley

Barley is an important cereal in Ireland, grown mainly on approximately 175,000 hectares each year. The vast majority (75%) of the national barley crop is sold as feed grain. About 22% of the national crop grown is classified as ‘malting barley’ and is used in the drinks industry for brewing, distilling and roasting. Approx 3% of the crop is grown for seed production each year.

Source: Central Statistics Office, Ireland. 2022

Symptoms

introduction

Barley leaf scald disease

Barley ‘Scald’ is an economically damaging fungal disease that is a global problem, causing significant yield and economical losses in barley feed and malting industries (Jonathan et al.,2021). The causal fungi is Rhynchosporium secalis.

Disease cycle

Source: Zhan et al., 2017.

INTRODUCTION

SUSCEPTIBILITY GENES

• Plants Susceptibilty genes are genes that are required for succesful pathogen infection

• The loss of function of S-genes disturb the compatible interaction and therefore lead to resistance.

• The resistance mediated by mutant alleles of S-genes have been reported in various plant species, and has been successfully applied to breeding for many years.

• The resistance has been reported to be durable.

01

Work Package one

'Identifying up to 10 bona fide S-genes in barley/leaf scald pathosystem'

WP1: tasks

task 1.1 LITERATURE STUDY

• Literature research of S-genes in other plants.

• Use information from model plants or other crops to identify homologs of S-genes from the availablce barley genomic database.

task 1.2: GENE EXPRESSION ANALYSIS

• Analyze Plant transcriptome data under infection of various pathogens.

• Protein to protein interaction e.g co-immunoprecipitation.

WP1: MATERIALS AND METHODS

IMMUNOPRECIPITATION Experiment

RNA SEQ Experiment

• Collect samples from infected and healthy barley plants.
• Extract total RNA from the samples.
• convert RNA to cDNA.
• Sequence and analyze sequence result.

Read more

02

Work Package TWO

DEVELOPING DYSFUNCTIONSL S-GENE USING CRISPR CAS 9

TASKs: Developing mutant lines using CRISPR-Cas9 gene editing method

Task 2.1:

• Design and construction of plasmid vector for loss of function mutant.

• Construct delivery in immature embryos using Agrobacterium mediated technique.

Task 2.2:

• Screening LOF mutants

CRISPR/Cas9 for targeted mutagenesis

Material and Method of WP2

Figure 1: pRGEB32-BAR Vector for the gene editing in maize using CRISPR-Cas9 (Hunter, 2021).

Task 2.1: Generating LOF mutants

• Design gRNAs: Snap gene, branchling, CRISPR-P etc.

• Obtain the sgRNA and the Cas9 protein.

• Select the plasmid for driving the gene editing for the generation of LOF mutants.

• Transform barley using Agrobacterium-mediated transformation.

• Select transformed cells and regenerate.

Material and Method of WP2

TASK 2.2: SCREENING OF MUTANTS

DNA extraction of mutants and Wild types, PCR analysis, gel electrophoresis, PCR quality assessment and sequencing.

• Sequence alignment and analysis using online tools e.g snapgene, clustal omega, NCBI.
• qPCR and or qRT-PCR.
• Protein expression analysis e.g western blot

Read more

03

Work package tHREE

Evaluate the resilience of the engineered barley lines to leaf scald and other relevant pathogens

AIM OF WP3.

This work package will be useful in:

• Evaluating the disease resillience of the mutated lines to barley leaf scald.
• Evaluate the agronomic and malting qualities of the new lines for any disease-plant growth trade off.

WP3: tasks

TASK 3.1:: GREEN HOUSE/ GROWTH CHAMBER EXPERIMENT

TASK 3:2: Imunity-growth trade-off EXPERIMENT

• Select Candidate mutants with no impact on critical impact on biochemical pathway from WP2.
• Choose an experimental design.
• Select pathogen(s) strain (s).
• Perform Disease innoculation.
• Carry out disease scoring.

• Conduct multi-location trials under controlled environment with different climate variables.

• Perform disease severity scoring.

• Measure economic and agronomic phenotypic traits.

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WP3: tasks

TASK 3.3 laboratory experiment

• Pathogen quantification.
• Measure activities of defense hormones e.g SA, JA in mutants
• Quantify secondary metabolites e.g phytoalexins.

wp3: Materials and methods

dISEASE INNOCULATION

dISEASE Diagnosis.

• Grow mutants and WT in a controlled environment.
• Inoculate the plants with a standardized spore suspension of Rhynchosporium commune.
• Maintain appropriate humidity and temperature conditions to promote infection.
• Follow the same procedure to innoculate other pathogens.

• Collect/record disease symptoms.
• Score disease severity.
• Collect plant samples.
• Use microscopy to identify pathogen
• isolate and grow pathogen in a culture media.
• Infect healthy plants with isolated pathogens and record symtoms.
• Analyze data.

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RESEARCH APPROACH

ADAPTIVE EXPERIENCE/SKILLS

WORK FLOW

Writing and research skills.

Literature Review

Experienced in conducting research experiments in Lab, green houses and field.

Conducting the Experiments.

Experienced in collecting phenotypic data. R programming skills.

Data collection and data analysis.

THANK YOU FOR THE ATTENTION

Questions?

Tip:

Interactivity is the key to capturing the interest and attention of your students. A genially is interactive because your students explore and engage with it.

work flow for rna seq Experinment.

Reverse transcritase quantitative pcr work flow

Check levels of SA/JA in LOF and WT

• Extract the two phytohormones from frozen samples.

• Perform quantification with high-performance liquid chromatography equipment coupled with a mass spectrometer.