Time-Course RNAseq Reveals Exserohilum turcicum Effectors and Pathogenicity Determinants.

Exserohilum turcicum (sexual stage Setosphaeria turcica) is the hemibiotrophic causal agent of northern leaf blight of maize and sorghum. This study aimed to identify the genes involved in host colonization during the biotrophic and necrotrophic phases of infection. It also aimed to identify race-specific differences in gene expression. RNAseq of maize seedlings inoculated with a race 13N or 23N E. turcicum isolate was conducted before inoculation and at 2, 5, 7, and 13 days post-inoculation (dpi). Biological replicates were pooled per time point for each race and sequenced. A bioinformatics pipeline was used to identify candidate effectors, and expression was validated for selected candidates. Fungal biomass was positively correlated with the percentages of E. turcicum reads mapped, which were low at early time points (2-7 dpi) with a significant increase at 13 dpi, indicating a lifestyle switch from biotrophy to necrotrophy between 7 and 13 dpi. AVRHt1 is the putative E. turcicum effector recognized by the maize resistance gene Ht1. Consistent with this, AVRHt1 was expressed in planta by race 23N, but transcripts were absent in race 13N. In addition, specific transposable elements were expressed in 23N only. Genes encoding the virulence-associated peptidases leupeptin-inhibiting protein 1 and fungalysin were expressed in planta. Transcriptional profiles of genes involved in secondary metabolite synthesis or cell wall degradation revealed the importance of these genes during late stages of infection (13 dpi). A total of 346 expressed candidate effectors were identified, including Ecp6 and proteins similar to the secreted in xylem (SIX) effectors common to formae speciales of Fusarium oxysporum, SIX13 and SIX5. Expression profiling of Ecp6 and SIX13-like indicated a peak in expression at 5 and 7 dpi compared to 2 and 13 dpi. Sequencing of SIX13-like from diverse isolates of E. turcicum revealed host-specific polymorphisms that were mostly non-synonymous, resulting in two groups of SIX13-like proteins that corresponded to the maize or sorghum origin of each isolate. This study suggests putative mechanisms whereby E. turcicum causes disease. Identification of the candidate effector SIX13-like is consistent with the infection mode of E. turcicum through the xylem of susceptible hosts.

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

Eucalyptus species are cultivated for forestry and are of economic importance. The fungal stem canker pathogen Chrysoporthe austroafricana causes disease of varying severity on E. grandis. The Eucalyptus grandis-Chrysoporthe austroafricana interaction has been established as a model system for studying Eucalyptus antifungal defence. Previous studies revealed that the phytohormone salicylic acid (SA) affects the levels of resistance in highly susceptible (ZG14) and moderately resistant (TAG5) clones. The aims of this study were to examine histochemical changes in response to wounding and inoculation as well as host responses at the protein level. The anatomy and histochemical changes induced by wounding and inoculation were similar between the clones, suggesting that anatomical differences do not underlie their different levels of resistance. Tyloses and gum-like substances were present after inoculation and wounding, but cell death occurred only after inoculation. Hyphae of C. austroafricana were observed inside dead and living cells, suggesting that the possibility of a hemibiotrophic interaction requires further investigation. Proteomics analysis revealed the possible involvement of proteins associated with cell death, SA signalling and systemic resistance. In combination with previous information, this study forms a basis for future functional characterisation of candidate genes involved in resistance of E. grandis to C. austroafricana.

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

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.