Austropuccinia psidii uses tetrapolar mating and produces meiotic spores in older infections on Eucalyptus grandis

•Investigation on mating genes STE3.2 and homeodomains indicateAustropuccinia psidiiuses tetrapolar mating, with implications for biosecurity.•Core meiotic genes in genomes of A. psidii, four species ofPuccinia, andSphaerophragmium acaciaeare conserved in rust fungi at family rank.•Expression of two meiotic and four mitotic genes ofA. psidiionE. grandiswere analysed over a 28-daytime course to validate that identified meiotic genes were upregulated in teliospores.•Significantly differential expression ofA. psidiistrain MF-1 meiotic genes indicate a temporal switch from production of uredinia (mitotic stage) to telia in the life cycle, which we hypothesize may be in response to leaf ageing. Austropuccinia psidii is the causal agent of myrtle rust, a fungal disease that infects over 480 species in the Myrtaceae. A. psidii is a biotrophic pathogen that reproduces sexually and asexually. Sexual reproduction has been previously shown on Syzygium jambos and little is known about its reproductive biology on other hosts or whether populations that were formerly structured by host range can outcross on universally susceptible hosts. We investigated if mating genes in three genomes of A. psidii were under selection as a proxy for whether different strains can reproduce sexually on a shared host. We examined three homologs of the STE3.2 gene, sequences of which were near-identical in the three genomes, and the homeodomain locus, which contained two alleles of two homeodomain genes in each genome. A. psidii likely uses tetrapolar mating. Pheromone/receptor loci were distal to homeodomain loci, and based on haplotypes of a phased assembly, mate compatibility is regulated by multiallelic HD genes and biallelic STE3.2 genes; the third homolog of STE3.2 (STE3.2-1) was present in both haplotypes, and our study supports hypotheses this gene does not regulate mate recognition. Populations of A. psidii formerly structured by host range could potentially outcross on universal hosts based on their related mating genes, however this hypothesis should remain theoretical given the implications for biosecurity. Additionally, we searched for core meiotic genes in genomes of A. psidii, four species of Puccinia, and Sphaerophragmium acaciae through comparative genomics based on 136 meiosis-related orthologous genes modeled from Mycosarcoma maydis. Meiotic genes are conserved in rust fungi at family rank. We analyzed the expression of two meiotic and four mitotic genes of A. psidii on