Damage caused by Phytophthora cinnamomi Rands remains an important concern on forest tree species. The pathogen causes root and collar rot, stem cankers, and dieback of various economically important Eucalyptus spp. In South Africa, susceptible cold tolerant Eucalyptus plantations have been affected by various Phytophthora spp. with P. cinnamomi considered one of the most virulent. The molecular basis of this compatible interaction is poorly understood. In this study, susceptible Eucalyptus nitens plants were stem inoculated with P. cinnamomi and tissue was harvested five days post inoculation. Dual RNA-sequencing, a technique which allows the concurrent detection of both pathogen and host transcripts during infection, was performed. Approximately 1% of the reads mapped to the draft genome of P. cinnamomi while 78% of the reads mapped to the Eucalyptus grandis genome. The highest expressed P. cinnamomi gene in planta was a putative crinkler effector (CRN1). Phylogenetic analysis indicated the high similarity of this P. cinnamomi CRN1 to that of Phytophthora infestans. Some CRN effectors are known to target host nuclei to suppress defense. In the host, over 1400 genes were significantly differentially expressed in comparison to mock inoculated trees, including suites of pathogenesis related (PR) genes. In particular, a PR-9 peroxidase gene with a high similarity to a Carica papaya PR-9 ortholog previously shown to be suppressed upon infection by Phytophthora palmivora was down-regulated two-fold. This PR-9 gene may represent a cross-species effector target during P. cinnamomi infection. This study identified pathogenicity factors, potential manipulation targets, and attempted host defense mechanisms activated by E. nitens that contributed to the susceptible outcome of the interaction.

RNA-sequencing technology has been widely adopted to investigate host responses during infection with pathogens. Dual RNA-sequencing (RNA-seq) allows the simultaneous capture of pathogen-specific transcripts during infection, providing a more complete view of the interaction. In this review, we focus on the design of dual RNAseq experiments and the application of downstream data analysis to gain biological insight into both sides of the interaction. Recent literature in this area demonstrates the power of the dual RNA-seq approach and shows that it is not limited to model systems where genomic resources are available. Sequencing costs continue to decrease and single cell transcriptomics is becoming more feasible. In combination with proteomics and metabolomics studies, these technological advances are likely to contribute to our understanding of the temporal and spatial aspects of dynamic plant-pathogen interactions. A dual approach in plantaThe interaction between plants and pathogens is an active and dynamic process that can be likened to a duel. Plants have complex defence mechanisms that can be rendered ineffective when pathogens interfere with one of the various processes required for host defence. These processes include penetration resistance, recognition by Pattern Recognition Receptors (PRRs), phytohormone signalling pathways, secretory pathways, secondary metabolite production, and plant cell death (Dou and Zhou, 2012). Until recently, transcriptomic approaches have been applied in the host and pathogen separately to obtain the gene expression profile of each organism and gain insight into infection biology or host defence mechanisms.RNA sequencing (RNA-seq) is a powerful technology that does not rely on any prior knowledge of transcripts and can generate vast quantities of data with much smaller costs involved than for older techniques such as microarrays (Pareek et al., 2011;Wilhelm and Landry, 2009). An advantage of RNA-seq in the field of plant-pathogen interactions is that both plant and pathogen transcripts can be detected simultaneously and accurately in the same sample. This tactic, known as dual RNAseq, in planta RNA-seq, simultaneous RNA-seq, or comparative RNA-seq, is a relatively new technique both in the plant and medical fields. In plants, it allows for the study of plant-pathogen interactions in herbaceous crops Kunjeti et al., 2012;Lowe et al., 2014) as well as trees (Hayden et al., 2014;Liang et al., 2014;Teixeira et al., 2014). This review outlines technical considerations for dual RNA-seq experiments, summarizes recent insights drawn from such approaches in plant-pathogen interactions, and provides an

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The rust pathogen, Puccinia psidii, was first detected in South Africa in 2013 on a single non-native ornamental Myrtus communis tree. This prompted surveys of the country to determine its geographic distribution and host range. Previously developed microsatellite markers where used to characterize P. psidii isolates collected from these surveys. In addition, artificial inoculation studies and field observations were used to evaluate the susceptibility of native Myrtaceae to infection by P. psidii. The pathogen was found on native Myrtaceae in isolated natural situations and it was also common on exotic Myrtaceae in nurseries and gardens. Marker analysis showed that a single genotype of the rust is present in South Africa and that this Unique Pucinnia psidii genotype in South Africa 2 is different to the so-called "pandemic" strain recorded in countries outside Brazil. It was found to have a broad distribution with collections as far as 1500 km apart. The data provide firm evidence for a single introduction of the pathogen from an as yet unkown source. Its wide distribution, particularly in relatively isolated natural areas, suggests that P. psidii has been present in South Africa for much longer than implied by its first detection in the country.

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Myrtle rust (Austropuccinia psidii) is an invasive species causing damage to Myrtaceae species in natural and managed ecosystems in many countries. To better understand myrtle rust epidemiology we studied latent period (LP) and ontogenic resistance in relation to temperature on three susceptible hosts (Metrosideros excelsa, Lophomyrtus bullata × L. obcordata and Syzygium jambos). The latent period curve was U‐shaped, with latent development >0 from between 8 and 10 °C, depending on the host, to 32 °C. Optimum range was 22–28 °C with minimum LP of 5–7 days. Peak spore production occurred over about 2 weeks, starting about 1 week after the LP ended. Some spore production continued for 1–2 months. Comparison of the LP data with field temperatures indicated that the uredinial stage of A. psidii can overwinter in the latent phase in temperate areas of New Zealand and southern Australia and, therefore, uredinial or telial reinfection is not required during winter. The LP information was used to correct the LP function in a New Zealand myrtle rust climatic risk model. The transition of emergent leaf and stem tissues in susceptible Myrtaceae genotypes from susceptible to immune (ontogenic resistance) was characterized in terms of uredinium density and LP. Onset of ontogenic resistance was closely linked to the degree of leaf expansion, with fully expanded leaves being immune to infection. Because ontogenic resistance restricts infection to periods when growth flushes occur, understanding it is crucial for explaining the seasonality of myrtle rust development in the natural environment.

Resistance to the pandemic strain of Austropuccinia psidii was identified in New Zealand provenance Leptospermum scoparium, Kunzea robusta, and K. linearis plants. Only 1 Metrosideros excelsa-resistant plant was found (of the 570 tested) and no resistant plants of either Lophomyrtus bullata or L. obcordata were found. Three types of resistance were identified in Leptospermum scoparium. The first two, a putative immune response and a hypersensitive response, are leaf resistance mechanisms found in other myrtaceous species while on the lateral and main stems a putative immune stem resistance was also observed. Both leaf and stem infection were found on K. robusta and K. linearis plants as well as branch tip dieback that developed on almost 50% of the plants. L. scoparium, K. robusta, and K. linearis are the first myrtaceous species where consistent infection of stems has been observed in artificial inoculation trials. This new finding and the first observation of significant branch tip dieback of plants of the two Kunzea spp. resulted in the development of two new myrtle rust disease severity assessment scales. Significant seed family and provenance effects were found in L. scoparium, K. robusta, and K. linearis: some families produced significantly more plants with leaf, stem, and (in Kunzea spp.) branch tip dieback resistance, and provenances provided different percentages of resistant families and plants. The distribution of the disease symptoms on plants from the same seed family, and between plants from different seed families, suggested that the leaf, stem, and branch tip dieback resistances were the result of independent disease resistance mechanisms.

The rust fungus Austropuccinia psidii has spread globally and naturalized in areas with na€ ıve species of Myrtaceae. Previous studies have revealed multiple strains of A. psidii within South America and two strains outside of its native range. The rust spreads by windborne mitotic urediniospores, which are the dominant spore stage. Teliospores and basidiospores of A. psidii are also formed; however, the biological role of these stages in the life cycle is unknown. Experiments presented here tested whether basidiospores of A. psidii could infect Syzygium jambos. The sori produced by infection with basidiospores were screened with five microsatellite markers to confirm whether they were a product of recombination. The findings showed that basidiospores of A. psidii could cause infection on species of Myrtaceae and the resulting sori were a product of recombination. This has important implications for programmes that breed for resistance to this aggressive pathogen in commercial eucalypt forestry.