Time-Course RNAseq Reveals Exserohilum turcicum Effectors and Pathogenicity Determinants

Abstract

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.

Diversity of Expression Types of Ht Genes Conferring Resistance in Maize to Exserohilum turcicum

“…The increase in XPE and XCE from 3 to 6 dpi indicates that between these time points, the pathogen is releasing virulence effectors. Transcriptional profiles showed that Ecp6 and SIX13-like proteins, similar to the secreted xylem effectors of Fusarium oxysporum, were overexpressed at 5 and 7 dpi (Human et al, 2020), which correlates to our findings in the histological studies and indicates that virulence effectors are being released at the time point before symptom expression (5-7 dpi).…”

“…xylem colonization started (Kotze et al, 2019). Moreover, gene effector candidates encoded a hybrid polyketide synthase:nonribosomal peptide synthetase (PKS:NRPS) (Wu et al, 2015), and virulence-associated peptidases leupeptin-inhibiting protein 1 fungalysin involved in the biosynthesis of secondary metabolites and cell wall degradation (Human et al, 2020). The increase in XPE and XCE from 3 to 6 dpi indicates that between these time points, the pathogen is releasing virulence effectors.…”

Benefits of maize resistance breeding and chemical control against northern leaf blight in smallholder farms in South Africa

“…Foliar symptoms are characterised by cigar-shaped lesions with pointed ends which are not constrained by vein margins, and therefore are wider and larger than GLS lesions. 16 The fungi causing GLS and NLB appear to exploit different ecological niches in maize leaves with C. zeina entering through stomata and proliferating between cells 17 , whereas E. turcicum colonises the xylem and uses this as a 'pathway' to move through the maize leaf 15 .…”

“…27 Indeed, KwaZulu-Natal had the worst drought in this season since 1921. 28 High humidity is required for optimal development of these diseases 12,16 , and therefore less disease is consistent with the drought season of 2015/2016. Furthermore, the 2014/2015 season was also subject to drought 28 , and therefore the ranking and significant disease differences between the seasons is consistent with rainfall levels.…”

Predector: an automated and combinative method for the predictive ranking of candidate effector proteins of fungal plant-pathogens

“…repeat-induced point mutation (RIP) 6,7 , as well as increased rates of chromosome structure rearrangement 8,9 and lateral gene transfer 10 . Consequently, fungal effectors are highly diverse in sequence and function, and much effector candidate discovery is performed using experimental techniques such as phenotype association and comparative genomics [11][12][13][14] , transcriptomics [15][16][17] , proteomics 18,19 and GWAS 20,21 . There are, however, some protein characteristics -i.e.…”