Exploring Resistance Gene Diversity and Evolution in the Genomes of Diverse Myrtaceae Species

Abstract

<p>Myrtaceae species are the most significant family of economic and ecological importance in Australia and are considered dominant in many diverse ecosystems. However, many are threatened by co-evolved and exotic diseases, including myrtle rust infection caused by <i>Austropuccinia psidii</i>. Intracellular nucleotide-binding sites and leucine-rich repeat (NBS-LRR) receptors play an essential role in plant response to biotic stresses. To investigate variation in phenotypic responses and identify the diversity and evolution of disease resistance gene family to facilitate future functional resistance gene (<i>R</i> gene) isolation in Myrtaceae, I screened diverse families of <i>Eucalyptus globulus</i> against <i>A. psidii</i> and conducted comprehensive annotation of NBS-encoding gene repertoires from long-read genome assembly of 21 species belonging to the Eucalypteae tribe of Myrtaceae. The genomes were sequenced using Oxford Nanopore Technology (ONT). </p> <p>From the annotated genomes, I identified over 55,536 NBS-encoding genes among 885,078 protein-coding gene models, of which 38,406 belong to the common TNL, CNL and RNL gene classes. Three-quarters of the annotated NBS-encoding genes were found in clusters. All species contained genes present in pairs of head-to-head orientation, suggesting the potential presence of sensor-executer genes that function in immune signalling. In this analysis, I examined 233 unique non-canonical domains (NCDs) integrated into the NBS encoding genes, with varying frequencies of occurrence among the species.</p> <p>Characterisation of the identified NCDs fused to the NBS-encoding genes in terms of diversity within and between species and gene classes shows that the type of NCDs and their integration frequency is highly varied among the species and the gene classes. Only a quarter of the identified NCDs were found in many analysed genomes. In contrast, the remaining three-quarters were rare among the species, indicating continued independent integration and <i>R</i> gene diversification. From the prevalent NCD domains, DNA, Ca2+ and carbohydrate binding domains, including zf-BED family, Jacalin, Calmodulin-binding and Protein kinase domains are most abundant across all species and gene classes. It was examined that Jacalin domain is highly expanded across all species, being integrated in C-terminal to the NBS genes that encode an N-terminal TIR domain (TN) but lacked leucine-rich repeat (LRR) domain, and rarely into the N-domain that lacks both N-and-C- terminal domain (N). Overlaying the NCDs over the phylogenetic tree of NCD containing proteins NB-ARC region reveals Jacalin domain from TN and N were clustered in a large single clade. Hence, I suggest that these domains have evolved with distinct characteristics likely due to duplication from ancestral integration. Jacalin domain integrated into TN genes in a repeated fashion as LRR does in typical NLR genes, suggest that the Jacalin domain might have replaced the LRR domains for pathogen sensing. I describe this potential new class of <i>R</i> genes as TNJ (TIR-NBS-Jacalin) genes. </p> <p>Phylogenetic analysis of TNJ from the broader representative species of Myrtaceae showed that TNJ formed a monophyletic clade between the TNL clades and evolved with the conservation of necessary motifs in its NBS domain and essential amino-acid residues including the catalytic glutamic acid (E) in its TIR domains. Shannon entropy and positive selection analyses show that most hyper-variable sites and residues that underwent diversifying selection have been clustered in the Jacalin region of TNJ, suggesting that surfaces of the Jacalin domain would potentially harbour a cluster of pathogen recognition specificity determining residues. These findings suggest that TNJ would potentially be a new class of <i>R</i> gene family in Myrtaceae with Jacalin as a replacement for LRR. The large number of NBS-encoding gene repertoire identified in this finding suggests the pathogen recognition potential possessed in Myrtaceae and the importance of such documentation in a future comparative and evolutionary study in flowering plants.</p> <p>The phenotypic screening shows high genetic variability among individuals with resistance responses manifested as a non-symptom or hypersensitive response (HR) to <i>A. psidii</i>. Individuals within families exhibited highly resistant and highly susceptible responses are a potential for targeted resistance sequencing to narrow down the identification of presence-absence markers variation influencing disease resistance for candidate <i>NLR</i> genes associated to the major resistance loci.</p>