The response of Eucalyptus grandis to drought stress, rewatering and combined stress involving Chrysoporthe austroafricana infection

Abstract

Forest trees encounter sequential or simultaneous combinations of several biotic and abiotic stresses recurring throughout their long life cycle. This could be exacerbated by climate change, which intensifies the severity, frequency, and duration of abiotic stressors such as drought, as well as international trade, which increases the global movement of pests and pathogens. Such combinations of biotic and abiotic stresses could interact either synergistically or antagonistically, resulting in more or less damage to trees compared to when they are exposed to individual stresses although there have been more observations linking combined stress conditions to increases in disease and insect pest outbreaks, massive tree mortality, and reduction in tree growth all of which lead to significant ecological and economic losses. Nevertheless, previous studies have primarily focused on plant responses to single stresses, often under controlled conditions. While these studies have contributed immensely to the mechanistic understanding of resistance to individual stresses, the response of plants to stress combinations, which they often face in the field conditions, have been suggested to be different from their responses to individual stresses. The Eucalyptus grandis - Chrysoporthe austroafricana interaction, a model pathosystem for studying forest tree diseases, could be expanded to investigate combined stress responses. Previous studies employing transcriptomics, proteomics, and metabolomics techniques have suggested that the phytohormone signaling pathways involving salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and gibberellic acid (GA) are among the key players conferring moderate resistance or facilitating susceptibility to the single stress of C. austroafricana infection in E. grandis. However, the molecular mechanisms underlying the response to and recovery from the single stress of drought have not been investigated in E. grandis. Thus, we first set up greenhouse experiments involving drought stress and subsequent rewatering where we collected stem samples for RNA sequencing (RNA-seq) analysis. Measurements of the stomatal conductance of leaves suggested that the trees gradually close their stomata during drought stress and reopen them relatively quickly upon rewatering. The RNA-seq data revealed the importance of the abscisic acid (ABA) and reactive oxygen species (ROS) signaling pathways as well as non-structural carbohydrate metabolism during drought stress. Upon rewatering, the ABA and ROS signaling pathways were repressed while primary metabolism and ROS catabolic processes were induced. Additionally, genes related to the SA and JA/ET signaling pathways were also downregulated during recovery from drought. Furthermore, using co-expression network analysis, we identified key genes that potentially govern the responses of E. grandis to drought stress and rewatering. To understand how the responses to the individual stresses of drought and C. austroafricana infection change under combined stress conditions, we conducted a greenhouse experiment involving the combination of pathogen infection with drought stress and subsequent rewatering. Stem lesion length measurements suggested that mild drought stress at the early time points could have predisposed the plants to pathogen attack while more severe drought at the later timepoints could have affected the pathogens as well as the hosts. Co-expression network analysis using RNA-seq data from the current combined stress experiment and previous single stress studies identified key genes whose expression patterns were altered in the moderately resistant genotype under combined stress conditions and resembled those in the more susceptible genotype under the single stress of pathogen infection. These included genes related to the SA, JA/ET, and GA biosynthesis and signaling pathways and support the observed drought-induced susceptibility to the pathogen. To compare the molecular mechanisms of recovery from drought stress under single and combined stress conditions, we investigated the transcriptomic profile of E. grandis during rewatering following exposure to the single and combined stresses of drought and pathogen infection. We found that rewatering following exposure to combined stress triggers distinct transcriptomic changes that are different from both the delayed responses to the single stress of pathogen infection and recovery from the single stress of drought. This study showed that the trees prioritized stress responses, mainly involving the JA/ET and ABA-independent signaling pathways, during recovery from combined stress conditions at the expense of growth and carbon storage. However, we found transcriptomic evidence suggesting carbon starvation during recovery from combined stress, which may be related to a decrease in photosynthetic rate as the downregulation of genes related to photosynthesis suggests and a potential increase in carbon demand, which can be attributed to the activation of stress response pathways. The changes in the abiotic environment, as in the case of drought stress and rewatering, could also affect the pathogen. To understand the effect of drought stress and subsequent rewatering in the pathogen, we investigated the changes in the in planta fungal transcriptome under these conditions. We found evidence suggesting that the pathogen may have maintained its metabolic activity and strengthened its cell wall under drought stress, implying prioritization of survival in the drought stressed host environment. However, some pathogenicity-related genes, including some cell wall degrading enzymes, were downregulated, suggesting that the increased disease progression under mild drought stress could be due to increased host susceptibility rather than enhanced virulence. Alternatively, the pathogen may retain the ability to infect the now-susceptible host through altered strategies, at least under mild drought stress. Upon rewatering, some genes related to fungal metabolism were downregulated, which suggests that the pathogen could take advantage of the changes in the host, and thus, could contribute to carbon starvation in the plants. Overall, this study revealed the molecular changes in both the host and the pathogen during drought stress and subsequent rewatering, shedding light on the molecular mechanisms underlying the complex tree-pathogen interactions during episodes of weather extremes and recovery periods.