The ectomycorrhizal fungus Pisolithus microcarpusencodes a microRNA involved in cross-kingdom gene silencing during symbiosis

Small RNAs (sRNAs) are known to regulate pathogenic plant-microbe interactions. Emerging evidence from the study of these model systems suggests that microRNAs (miRNAs) can be translocated between microbes and plants to facilitate symbiosis. The roles of sRNAs in mutualistic mycorrhizal fungal interactions, however, are largely unknown. In this study, we characterized miRNAs encoded by the ectomycorrhizal fungus Pisolithus microcarpus and investigated their expression during mutualistic interaction with Eucalyptus grandis. Using sRNA sequencing data and in situ miRNA detection, a novel fungal miRNA, Pmic_miR-8, was found to be transported into E. grandis roots after interaction with P. microcarpus. Further characterization experiments demonstrate that inhibition of Pmic_miR-8 negatively impacts the maintenance of mycorrhizal roots in E. grandis, while supplementation of Pmic_miR-8 led to deeper integration of the fungus into plant tissues. Target prediction and experimental testing suggest that Pmic_miR-8 may target the host NB-ARC domain containing transcripts, suggesting a potential role for this miRNA in subverting host signaling to stabilize the symbiotic interaction. Altogether, we provide evidence of previously undescribed cross-kingdom sRNA transfer from ectomycorrhizal fungi to plant roots, shedding light onto the involvement of miRNAs during the developmental process of mutualistic symbioses

The ectomycorrhizal fungus Pisolithus microcarpus encodes a microRNA involved in cross-kingdom gene silencing during symbiosis.

Small RNAs (sRNAs) are known to regulate pathogenic plant-microbe interactions. Emerging evidence from the study of these model systems suggests that microRNAs (miRNAs) can be translocated between microbes and plants to facilitate symbiosis. The roles of sRNAs in mutualistic mycorrhizal fungal interactions, however, are largely unknown. In this study, we characterized miRNAs encoded by the ectomycorrhizal fungus Pisolithus microcarpus and investigated their expression during mutualistic interaction with Eucalyptus grandis. Using sRNA sequencing data and in situ miRNA detection, a novel fungal miRNA, Pmic_miR-8, was found to be transported into E. grandis roots after interaction with P. microcarpus Further characterization experiments demonstrate that inhibition of Pmic_miR-8 negatively impacts the maintenance of mycorrhizal roots in E. grandis, while supplementation of Pmic_miR-8 led to deeper integration of the fungus into plant tissues. Target prediction and experimental testing suggest that Pmic_miR-8 may target the host NB-ARC domain containing transcripts, suggesting a potential role for this miRNA in subverting host signaling to stabilize the symbiotic interaction. Altogether, we provide evidence of previously undescribed cross-kingdom sRNA transfer from ectomycorrhizal fungi to plant roots, shedding light onto the involvement of miRNAs during the developmental process of mutualistic symbioses

All‐in‐one Xylella detection and identification: A nanopore sequencing‐compatible conventional PCR

Xylella fastidiosa is a plant-pathogenic bacterium that poses a serious threat to the production of economically important plant species including grapes, almonds, olives and a broad range of amenity plants, causing significant economic losses worldwide. While multiple molecular detection assays have been developed for X. fastidiosa, there is a lack of molecular tools available for detection and differentiation of the closely related pear pathogen, Xylella taiwanensis. In this study, we present a novel conventional PCR assay with primers that can amplify both Xylella species. The amplified product could be sequenced and used for discrimination between the two species and the subspecies within the fastidiosa species. This PCR assay was designed using a genome-informed approach to target the ComEC/Rec2 gene of both Xylella species, ensuring a higher specificity than other previously developed PCR assays. A test performance study across five national plant diagnostic laboratories in Australia and New Zealand demonstrated this assay's high sensitivity and specificity to all known species and subspecies within the Xylella genus. This PCR assay can be used for Xylella identification at the species and subspecies level and is compatible with Sanger sequencing and nanopore sequencing for rapid turnaround time. The newly developed conventional PCR assay presented here offers rapid detection and accurate identification of both Xylella species from plant, insect vector or bacterial samples, enabling timely implementation of biosecurity measures or disease management responses

Untangling the effect of roots and mutualistic ectomycorrhizal fungi on soil metabolite profiles under ambient and elevated carbon dioxide

Metabolites in soil play an important role in regulating plant-microbe interactions and, thereby, plant performance. Biotic factors such as root exudation and microbial activity or abiotic factors such as the concentration of atmospheric carbon dioxide (CO2) can drive both quantitative and qualitative changes in soil metabolite profiles. While the impact of these factors, either in isolation or in combination, are underexplored in soil systems due to technical challenges, recent technological advances have enabled these hurdles to be overcome. Given the key role that mutualistic ectomycorrhizal (ECM) fungi play in forest soils through their symbiotic interaction with trees, and the foreseen changes in forest dynamics with climate change, we investigated the effect of the Eucalyptus grandis-Pisolithus albus (plant host-fungus) association on soil metabolite profiles under ambient and elevated CO2 conditions (aCO2 and eCO2). We found that significant metabolite enrichment predominately occurred in the rhizosphere where a strong effect by ECM fungus was also observed. Specific ECM fungus-induced metabolites were enriched concurrently with an increased host plant root:shoot ratio, suggesting that the influence of ECM fungus on rhizosphere metabolite profiles may impact plant growth. Strikingly, however, we found no observable differences in soil metabolite profiles between the aCO2 and eCO2 conditions, which may be due to nutrient limitation given the low level of nutrients found in typical eucalyptus forest soils. Overall, our findings increase our understanding of soil metabolic processes at the symbiotic plant-microbe interface under current and future atmospheric CO2 scenarios

Vertical and horizontal gene transfer shaped plant colonization and biomass degradation in the fungal genus Armillaria

The fungal genus Armillaria contains necrotrophic pathogens and some of the largest terrestrial organisms that cause tremendous losses in diverse ecosystems, yet how they evolved pathogenicity in a clade of dominantly non-pathogenic wood degraders remains elusive. Here we show that Armillaria species, in addition to gene duplications and de novo gene origins, acquired at least 1,025 genes via 124 horizontal gene transfer events, primarily from Ascomycota. Horizontal gene transfer might have afected plant biomass degrading and virulence abilities of Armillaria, and provides an explanation for their unusual, soft rot-like wood decay strategy. Combined multi-species expression data revealed extensive regulation of horizontally acquired and wood-decay related genes, putative virulence factors and two novel conserved pathogenicity-induced small secreted proteins, which induced necrosis in planta. Overall, this study details how evolution knitted together horizontally and vertically inherited genes in complex adaptive traits of plant biomass degradation and pathogenicity in important fungal pathogens

Nitrogen fertilization differentially affects the symbiotic capacity of two co-occurring ectomycorrhizal species

Forest trees rely on ectomycorrhizal (ECM) fungi to obtain growth-limiting nutrients. While addition of nitrogen (N) has the potential to disrupt these critical relationships, there is conflicting evidence as to the mechanism by which ECM:host mutualism may be affected. We evaluated how N fertilization altered host interactions and gene transcription between Eucalyptus grandis and Pisolithus microcarpus or Pisolithus albus, two closely related ECM species that typically co-occur within the same ecosystem. Our investigation demonstrated species-specific responses to elevated N: P. microcarpus maintained its ability to transport microbially sourced N to its host but had a reduced ability to penetrate into root tissues, while P. albus maintained its colonization ability but reduced delivery of N to its host. Transcriptomic analysis suggests that regulation of different suites of N-transporters may be responsible for these species-specific differences. In addition to Ndependent responses, we were also able to define a conserved ‘core’ transcriptomic response of Eucalyptus grandis to mycorrhization that was independent of abiotic conditions. Our results demonstrate that even between closely related ECM species, responses to N fertilization can vary considerably, suggesting that a better understanding of the breadth and mechanisms of their responses is needed to support forest ecosystems into the future

Species-level identity of Pisolithus influences soil phosphorus availability for host plants and is moderated by nitrogen status, but not CO2

Trees are dependent on the activity of soil microorganisms, including mutualistic ectomycorrhizal (ECM) fungi and soil-dwelling bacteria, for access to phosphorus (P). While P is a key limiting nutrient in temperate and other forest ecosystems, our understanding of the contributions of ECM fungi to plant P nutrition and cycling are unclear. Further complicating our understanding of these processes are the combined effects of fungal species and future climate scenarios and soil nutrient availability. In this study we characterised how the ECM fungi Pisolithus albus and Pisolithus microcarpus influenced the amount of plant-available P in soils and plant P content of Eucalyptus grandis. We explored how these fungi influence P cycling by studying their relative P-solubilising and P-mineralising abilities and examining their impact on P cycling gene abundance in the soil bacterial community. These were investigated under different levels of nitrogen addition and atmospheric CO2 to understand how these processes may be impacted by future anthropogenic and climactic change. While inoculation with either P. albus or P. microcarpus resulted in an increase in plant-available P in soil, the amount of plant-available P mobilised was species-specific. This observation was supported by differences in the in vitro P-mobilising abilities of the two fungi. P. albus and P. microcarpus also favoured bacterial communities characterised by a greater abundance of glucose dehydrogenase gene copies for inorganic P solubilisation, complementing the strengths of the ECM fungi in organic P mineralisation and suggesting a distinction in the roles of ECM fungi and bacteria in P cycling. Furthermore, both nitrogen and CO2 levels impacted these P cycling outcomes, often in a species-specific manner. Our findings expand the current understanding of P cycling between forest trees, ECM fungi and soil bacteria, and have important implications for estimations of future anthropogenic impacts on forest ecosystems

Abscisic acid supports colonization of Eucalyptus grandis roots by the mutualistic ectomycorrhizal fungus Pisolithus microcarpus

The pathways regulated in ectomycorrhizal (EcM) plant hosts during the establishment of symbiosis are not as well understood when compared to the functional stages of this mutualistic interaction. Our study used the EcM host Eucalyptus grandis to elucidate symbiosis-regulated pathways across the three phases of this interaction. Using a combination of RNA sequencing and metabolomics we studied both stage-specific and core responses of E. grandis during colonization by Pisolithus microcarpus. Using exogenous manipulation of the abscisic acid (ABA), we studied the role of this pathway during symbiosis establishment. Despite the mutualistic nature of this symbiosis, a large number of disease signalling TIRNBS-LRR genes were induced. The transcriptional regulation in E. grandis was found to be dynamic across colonization with a small core of genes consistently regulated at all stages. Genes associated to the carotenoid/ABA pathway were found within this core and ABA concentrations increased during fungal integration into the root. Supplementation of ABA led to improved accommodation of P. microcarpus into E. grandis roots. The carotenoid pathway is a core response of an EcM host to its symbiont and highlights the need to understand the role of the stress hormone ABA in controlling host–EcM fungal interactions