Research suggests genomic markers may help control a fungal Myrtle Rust pathogen. By Sam Carr, QAA member and Director and Senior Consultant at Queensland’s Specialist Arbor Services.
Myrtle Rust (Austropuccinia psidii) is a biotrophic fungal pathogen that affects more than 480 species of Myrtaceous plants globally. It was first discovered in Australia in 2010, and given the lack of coevolution within the native populations of Myrtaceae, infection susceptibility was high and the pathogen spread rapidly. Within 12 months of emergence, containment of Myrtle Rust was deemed unachievable and current efforts to save susceptible trees aimed to focus on resistant genotypes and pathogen biological control.
The pathogen
Through genetic analysis, genetic markers confirm the A.psidii found in Australia originated from Brazil. Research using scanning electron microscopes examined how the rust fungus infects leaves. Results concluded there were no obvious differences in the development of infectious structures (urediniospores) of A.psidii, regardless of whether the plant was completely resistant, hypersensitive, or susceptible in the eucalyptus species studied.
Germinating urediniospores penetrate the leaf cuticle in susceptible specimens, but not in completely resistant varieties, indicating the resistance within specimens may lie within the genome responsible for morphological features of the leaf. Given the high profile of A.psidii and the impacts on ecology and economics it can have, scientific research into the disease has been extensive. Researchers agree the pathogen can be highly adaptable due to the transposable-rich genome (TRG).
TRGs are genomes with have highly mobile DNA sequences that can move to alternate positions within the highly susceptible species.
Historically, Myrtaceae occupied the ancient land of Gondwana. When Gondwana split, the trees in the Americas and Australia evolved into their own species. It is theorised these ancient trees may have co-evolved with the disease, resulting in some modern specimens demonstrating resistance. Theoretically, modern species could carry ancient or fossil genes deep within the conserved architecture of the genome. Research and genome-level analyses of molecular markers suggest the presence of these ‘R’ genes may be conserved within the genome of some resistant specimens. However, the specific gene, genomic region, or single-base variation of a detectable locus responsible for an ancestral resistance gene has yet to be identified and confirmed.
The pathway to resistance
Early Myrtle Rust research deliberately infected 23 clones of various eucalyptus species from five provenances with A.psidii. The results demonstrated neither provenance nor species equated to definite resistance or susceptibility. Varying levels of both resistance and susceptibility were noted during the phenotypical response, and no obvious indicators were drawn from the experiment. This helped direct the research to genetic testing on plants demonstrating phenotypical responses of resistance, and to compare those genomes with specimens with phenotypically susceptible traits, to attempt to identify the expression of different genes or loci responsible for the desired trait.
Genetic studies of the selected Myrtaceous species aim to clarify to the mode of resistance within these trees by examining variations in genetics at multiple genomic levels:
* Complete genome mapping;
* loci identification;
* identification of individual genes (functional DNA sequence); and
* Single Nucleotide Polymorphism (SNP) – a change of a single nucleotide in a sequence that helps map areas of interest, or a change in the genes due to a change in alleles within a suspected gene responsible for resistance.
Study
One method to aid in identification of such expressed genes is a genome-wide association study (GWAS).
A GWAS can test thousands of genetic variants within a chosen population, enabling differences within DNA sequences across specimens or populations to be identified and statistically associated with resistance. This enables genomic regions to be identified and candidate genes to be evaluated to help confirm pathogen resistance.
The GWAS found a commonality between phenotypes (observable expression of a trait) and genotypes (genetic makeup that produces that trait) suggesting specimens with improved resistance were linked to both loci and polymorphic genes (genes that vary due to changes in alleles).
Quantitative trait loci
Patterns of genes suspected of being responsible for a desirable trait, such as pathogen resistance, are known as Quantitative Trait Loci (QTLs). QTLs are the genomic regions consisting of multiple genes or multiple loci within the genome acting together that are suspected of being responsible for a trait. These areas within the genome have been mapped via Single Nucleotide Polymorphism (SNP)-based analysis.
Single-nucleotide polymorphism
SNPs are single-nucleotide differences within a DNA sequence. These changes in the DNA sequence may or may not influence the way a gene is expressed, but can be used to identify the QTLs, allowing evaluation of the genes present in those loci.
The research concluded there are the five major loci that may influence varying levels of rust resistance within Eucalyptus grandis, Eucalyptus globulus, and E obliqua. Given the consistent evidence these regions are linked to rust resistance within these species, the mapped regions can then be applied to, and compared with, new species to determine if they are present within related tree species.
Transcriptomics
Transcriptomics can be implemented to determine if candidate genes are functional during myrtle rust infection.
Transcriptomics is the process of measuring RNA molecules in tissue samples at a chosen time before and after infection. Due to the unstable nature of RNA, it must be converted to complementary DNA (cDNA). The cDNA is then sequenced using high throughput platforms, which allows comparison across samples.
These results are compared to a reference genome, other resistance specimens, and susceptible specimens. The loci and/or genes of interest can then be compared and analysed to determine if the genes within the QTLs exhibit differential expression. The rate of activation can also be monitored over specific time intervals, which can also indicate how resistant a specimen may be. This analysis helps recognise high-confidence candidate genes.
Alternative approach
Concurrent research of an alternative mode of protection using genome-driven science has been implemented to target the pathogen directly. This involved spraying double-strand RNA (dsRNA) onto leaves of susceptible tree species.
The dsRNA is created to invoke a defence response in the pathogen and intercept messenger RNA coding for infection methods such as haustoria development. Once bonded to the specific mRNA molecule, the dsRNA silences it, restricting the pathogen from infecting susceptible leaves. The dsRNA is specifically created to target a known gene of the pathogen and therefore will not affect the broader biome of the treatment area. The dsRNA can also be modified as the pathogen evolves, enabling active genes in new strains to be silenced faster than the development of new resistant trees.
Further work required
Tremendous effort has been invested in understanding myrtle rust resistance through genomic mapping, QTL analyses, and associated research. These approaches have highlighted several genomic regions likely to contain genes contributing to reduced disease severity; however, no study has yet isolated a single causal gene or locus, nor has any breeding program produced genotypes demonstrating complete, heritable resistance. Two theories of broad resistance mechanisms acting on rust resistance within the genome have been proposed: ancestrally conserved resistant loci and more recently adapted quantitative immune responses.
Current evidence cannot yet confirm whether resistance reflects an inherited mechanism present across the family, or whether it arises from multiple loci acting in combination through quantitative, polygenic resistance, or natural variations of alleles within the plant defence systems. Further comparative work across diverse Myrtaceae lineages is required to resolve whether these defence responses in resistant specimens exhibit a shared ancestral trait, independent multi-locus adaptations, or both acting independently or cohesively to increase resistance.
Recognising the genomic diversity within the Myrtaceae family, and the complexity of the interacting mechanisms that are yet to be confirmed for rust resistance, evidence suggests developing reliable resistant specimens will take time. Even if future research confirms stable resistance pathways and selective breeding programs succeed in producing more tolerant genotypes, the highly adaptable nature of Austropuccinia psidii raises concerns that the pathogen will eventually overcome these resistant trees due to the highly adaptable transposable genome of the pathogen.
Combining targeted dsRNA-based suppression of Austropuccinia psidii with continued genomic research to identify and validate resistance loci is essential for protecting ecologically and economically important Myrtaceae species. More genome mapping and functional validation are required before breeders can reliably produce genotypes with stable, heritable resistance.
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This article was the result of collating research from a variety of respected authors. Sam Carr would like to acknowledge the following:
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(Yong et al. 2021)
