Localization and Transcriptional Responses of Chrysoporthe austroafricana in Eucalyptus grandis Identify Putative Pathogenicity Factors

Abstract

Chrysoporthe austroafricana is a fungal pathogen that causes the development of stem cankers on susceptible Eucalyptus grandis trees. Clones of E. grandis that are partially resistant and highly susceptible have been identified based on the extent of lesion formation on the stem upon inoculation with C. austroafricana. These interactions have been used as a model pathosystem to enhance our understanding of interactions between pathogenic fungi and woody hosts, which may be different to herbaceous hosts. In previous research, transcriptomics of host responses in these two clones to C. austroafricana suggested roles for salicylic acid and gibberellic acid phytohormone signaling in defense. However, it is unclear how the pathogen infiltrates host tissue and which pathogenicity factors facilitate its spread in the two host genotypes. The aim of this study was to investigate these two aspects of the E. grandis–C. austroafricana interaction and to test the hypothesis that the pathogen possesses mechanisms to modulate the tree phytohormone-mediated defenses. Light microscopy showed that the pathogen occurred in most cell types and structures within infected E. grandis stem tissue. Notably, the fungus appeared to spread through the stem by penetrating cell wall pits. In order to understand the molecular interaction between these organisms and predict putative pathogenicity mechanisms of C. austroafricana, fungal gene expression was studied in vitro and in planta. Fungal genes associated with cell wall degradation, carbohydrate metabolism and phytohormone manipulation were expressed in planta by C. austroafricana. These genes could be involved in fungal spread by facilitating cell wall pit degradation and manipulating phytohormone mediated defense in each host environment, respectively. Specifically, the in planta expression of an ent-kaurene oxidase and salicylate hydroxylase in C. austroafricana suggests putative mechanisms by which the pathogen can modulate the phytohormone-mediated defenses of the host. These mechanisms have been reported in herbaceous plant–pathogen interactions, supporting the notion that these aspects of the interaction are similar in a woody species. This study highlights ent-kaurene oxidase and salicylate hydroxylase as candidates for further functional characterization.

Evidence for salicylic acid signalling and histological changes in the defence response of Eucalyptus grandis to Chrysoporthe austroafricana

“…Lesion formation similar to previous reports357 was observed in all inoculated plants. After 42 days, the wounded plants had formed new callus tissue that occluded all or most of the original wound (Supplementary Figure S1A).…”

“…While the location and spread of C. austroafricana during infection of E. grandis have been described7, the responses of E. grandis to infection with C. austroafricana have not been studied at the microscopic level. This information could provide clues about the physical changes in the host during wounding and infection, some of which could contribute to resistance, and about pathogen lifestyle.…”

“…Callus was absent from the inoculated plants. Wilting and death occurred in some of the inoculated plants by 42 dpi7.…”

“…The initial observations detailed here show that there are no drastic differences in the histological responses to infection between the two clones. However, since the rate of lesion development differs between these clones357 and cell damage coincides with the appearance of the lesion, it is possible that the rate of cell damage is different in these clones.…”

“…To understand the role of the pathogen in the interaction, the C. austroafricana draft genome was used to perform a dual RNA-Seq analysis7. Several putative virulence genes were identified, and the results suggested possible manipulation of SA and gibberellic acid (GA) signalling as well as plant cell wall degradation by the pathogen.…”