Does fungal infection increase the palatability of oilseed rape to insects?

BACKGROUND: Multiple and simultaneous attacks by pathogens and insect pests frequently occur in nature. Plants respond to biotic stresses by activating distinct defense mechanisms, but little is known about how plants cope with multiple stresses. The focus of this study was the combined interaction of fungal infection caused by Leptosphaeria maculans (synonym Plenodomus lingam) and arthropod infestation by the diamondback moth (Plutella xylostella) in oilseed rape (Brassica napus). We hypothesized that infection by the fungal pathogen L. maculans could alter oilseed rape palatability to P. xylostella-chewing caterpillars. Feeding preference tests were complemented with analyses of defense gene transcription, and levels of glucosinolates (GLSs) and volatile organic compounds (VOCs) in L. maculans-inoculated and non-inoculated (control) leaves to determine possible causes of larval choice. RESULTS: Caterpillars preferred true leaves to cotyledons, hence true leaves were used for further experiments. True leaves inoculated with L. maculans were more palatable to caterpillars over control leaves during the early stage of infection at 3 days post inoculation (dpi), but this preference disappeared in the later stages of infection at 7 dpi. In parallel, genes involved in the salicylic acid and ethylene pathways were up-regulated in L. maculans-inoculated leaves at 3 and 7 dpi; L. maculans increased the level of total aliphatic GLSs, specifically glucobrassicanapin, and decreased the level of glucoiberin at 3 dpi and altered the content of specific VOCs. A group of 55 VOCs with the highest variability between treatments was identified. CONCLUSION: We suggest that the P. xylostella preference for L. maculans-inoculated leaves in the early stage of disease development could be caused by the underlying mechanisms leading to changes in metabolic composition. Further research should pinpoint the compounds responsible for driving larval preference and evaluate whether the behavior of the adult moths, i.e. the stage that makes the first choice regarding host plant selection in field conditions, correlates with our results on larval host acceptance. © 2024 Society of Chemical Industry.

Regulation of effector gene expression as concerted waves in Leptosphaeria maculans: a two-player game.

Effector genes, encoding molecules involved in disease establishment, are concertedly expressed throughout the lifecycle of plant-pathogenic fungi. However, little is known about how effector gene expression is regulated. Since many effector genes are located in repeat-rich regions, the role of chromatin remodeling in their regulation was recently investigated, notably establishing that the repressive histone modification H3K9me3, deposited by KMT1, was involved in several fungal species including Leptosphaeria maculans. Nevertheless, previous data suggest that a second regulatory layer, probably involving a specific transcription factor (TF), might be required. In L. maculans, a Dothideomycete causing stem canker of oilseed rape, we identified the ortholog of Pf2, a TF belonging to the Zn2Cys6 fungal-specific family, and described as essential for pathogenicity and effector gene expression. We investigated its role together with KMT1, by inactivating and over-expressing LmPf2 in a wild-type strain and a ∆kmt1 mutant. Functional analyses of the corresponding transformants highlighted an essential role of LmPf2 in the establishment of pathogenesis and we found a major effect of LmPf2 on the induction of effector gene expression once KMT1 repression is lifted. Our results show, for the first time, a dual control of effector gene expression.

Leptosphaeria maculans isolates with variations in AvrLm1 and AvrLm4 effector genes induce differences in defence responses but not in resistance phenotypes in cultivars carrying the Rlm7 gene.

BACKGROUND: The phoma stem canker pathogen Leptosphaeria maculans is one of the most widespread and devastating pathogens of oilseed rape (Brassica napus) in the world. Pathogen colonization is stopped by an interaction of a pathogen Avr effector gene with the corresponding host resistance (R) gene. While molecular mechanisms of this gene-for-gene interaction are being elucidated, understanding of effector function remains limited. The purpose of this study was to determine the action of L. maculans effector (AvrLm) genes on incompatible interactions triggered by B. napus noncorresponding R (Rlm) genes. Specifically, effects of AvrLm4-7 and AvrLm1 on Rlm7-mediated resistance were studied. RESULTS: Although there was no major effect on symptom expression, induction of defence genes (e.g. PR1) and accumulation of reactive oxygen species was reduced when B. napus cv. Excel carrying Rlm7 was challenged with a L. maculans isolate containing AvrLm1 and a point mutation in AvrLm4-7 (AvrLm1, avrLm4-AvrLm7) compared to an isolate lacking AvrLm1 (avrLm1, AvrLm4-AvrLm7). AvrLm7-containing isolates, isogenic for presence or absence of AvrLm1, elicited similar symptoms on hosts with or without Rlm7, confirming results obtained with more genetically diverse isolates. CONCLUSION: Careful phenotypic examination of isogenic L. maculans isolates and B. napus introgression lines demonstrated a lack of effect of AvrLm1 on Rlm7-mediated resistance despite an apparent alteration of the Rlm7-dependent defence response using more diverse fungal isolates with differences in AvrLm1 and AvrLm4. As deployment of Rlm7 resistance in crop cultivars increases, other effectors need to be monitored because they may alter the predominance of AvrLm7. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Large-scale population survey of Leptosphaeria maculans in France highlights both on-going breakdowns and potentially effective resistance genes in oilseed rape.

BACKGROUND: Leptosphaeria maculans, the cause of stem canker of oilseed rape, develops gene-for-gene interactions with its host and shows a high evolutionary potential to 'break down' novel resistance genes (R, Rlm) deployed in cultivars over large areas. For optimal management of R genes, updated knowledge of the population structure of the pathogen is needed. In France, large-scale surveys have been done at 10-year intervals since 2000. Here we report the characterization of a large L. maculans population collected in France in 2019-2020. RESULTS: A total of 844 isolates were collected from 11 sites in ten French departments and were phenotyped for their virulence against nine Brassica napus R genes. All isolates were virulent toward Rlm2 and Rlm9. Very few isolates were avirulent on Rlm1 (1.8%) and Rlm4 (0.6%). Avirulent isolates toward Rlm7 ('AvrLm7') varied from 67% to 11.3%, depending on the site sampled, illustrating the ongoing breakdown of Rlm7. The decrease of AvrLm7 isolates (29.2% at the national level) compared to the 2010 survey (96.5%) was accompanied by an increase of avirulent isolates on Rlm3 (0% in 2010; 54% in 2019-2020). However, virulent isolates on both Rlm3 and Rlm7, previously rarely detected, were found in all sites with a frequency of 17.3%. Finally, most or all isolates were avirulent on Rlm11 (96.1%), LepR2 (RlmS, 99.8%), and Rlm6 (100%), suggesting these three genes still effectively control the disease. CONCLUSION: These data will help guide strategies for breeding and deploying resistant oilseed rape varieties against L. maculans in France. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Co-inoculation timing affects the interspecific interactions between phoma stem canker pathogens Leptosphaeria maculans and Leptosphaeria biglobosa.

BACKGROUND: Phoma stem canker is an economically important disease of oilseed rape, caused by two co-existing fungal pathogen species, Leptosphaeria maculans (Plenodomus lingam) and Leptosphaeria biglobosa (Plenodomus biglobosus). Leptosphaeria maculans produces a phytotoxin called sirodesmin PL. Our previous work showed that L. biglobosa has an antagonistic effect on the production of sirodesmin PL if it is simultaneously co-inoculated with L. maculans. However, the effects of sequential co-inoculation on interspecific interactions between the two pathogens are not understood. RESULTS: The interactions between L. maculans and L. biglobosa were investigated in liquid culture by inoculation with L. maculans first, followed by L. biglobosa sequentially at 1, 3, 5 or 7 days later and vice versa; the controls were inoculated with L. maculans only, L. biglobosa only, or L. maculans and L. biglobosa simultaneously. The results showed that L. biglobosa inhibited the growth of L. maculans, the production of both sirodesmin PL and its precursors if L. biglobosa was inoculated before, or simultaneously with, L. maculans. However, the antagonistic effects of L. biglobosa were lost if it was co-inoculated 5 or 7 days after L. maculans. CONCLUSION: For the first time, the results of this study provided evidence that the timing when L. maculans and L. biglobosa meet significantly influences the outcome of the interspecific competition between them. Leptosphaeria biglobosa can inhibit the production of sirodesmin PL and the growth of L. maculans if it is inoculated before L. maculans or less than 3 days after L. maculans in liquid culture. There is a need to further investigate the timing of co-inoculation on interactions between L. maculans and L. biglobosa in their host plants for improving the control of phoma stem canker. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Efficient qPCR estimation and discrimination of airborne inoculum of Leptosphaeria maculans and L. biglobosa, the causal organisms of phoma leaf spotting and stem canker of oilseed rape.

BACKGROUND: Detection of the inoculum of phytopathogens greatly assists in the management of diseases, but is difficult for pathogens with airborne fungal propagules. Here, we present experiments to determine the abundance and distribution frequencies of the ascospores of Leptosphaeria (Plenodomus) species that were collected on the tapes of volumetric Hirst-type traps near oilseed rape fields in Poznan, Poland and Harpenden, UK. Fungal detection and species discrimination were achieved using a SYBR-Green quantitative polymerase chain reaction (qPCR) with two different pairs of primers previously reported to differentiate Leptosphaeria maculans (Plenodomus lingam) or L. biglobosa (P. biglobosus). RESULTS: Detection was successful even at fewer than five spores per m3 of air. The primer pairs differed in the correlation coefficients obtained between DNA yields and the daily abundance of ascospores that were quantified by microscopy on duplicate halves of the spore trap tapes. Important differences in the specificity and sensitivity of the published SYBR-Green assays were also found, indicating that the Liu primers did not detect L. biglobosa subclade 'canadensis', whereas the Mahuku primers detected L. biglobosa subclade 'canadensis' and also the closely related Plenodomus dezfulensis. CONCLUSIONS: Comparisons confirmed that application of qPCR assays to spore trap samples can be used for the early detection, discrimination and quantification of aerially dispersed L. maculans and L. biglobosa propagules before leaf spot symptoms are visible in winter oilseed rape fields. The specificity of the primers must be taken into consideration because the final result will greatly depend on the local population of the pathogen. © 2023 Society of Chemical Industry.

Development of a Duplex qPCR System for Detection and Quantification of the Two Canola Blackleg Pathogens Leptosphaeria biglobosa and L. maculans.

Two probe-based qPCR systems, namely P-Lb and P-Lm, specific to the canola blackleg pathogens Leptosphaeria biglobosa and L. maculans, respectively, were developed, and their efficiencies were tested. Each of the two systems targets a single-copy gene exclusively present in the corresponding species. The specificities of the two systems on the species level and their ubiquities on the subspecies level were confirmed by in silico sequence analyses and testing on L. biglobosa (17 strains), L. maculans (10 strains), and other plant pathogens (31 species). For sensitivities, the two systems were tested on synthesized DNA fragments (gBlock) of the targeted regions, from which a standard curve was generated for each system. In addition, standard curves were also generated on gBlocks for duplex qPCR in which the two systems were used in the same reaction. The two systems were further tested in both singleplex and duplex qPCR on DNA samples extracted from fungal spores, inoculated canola cotyledons, and naturally infected canola stubble samples collected from commercial fields. Our data indicated that the two systems are specific to L. biglobosa and L. maculans, respectively, and one reaction could detect as few as 200 spores of either species. When used in duplex qPCR on DNA samples with various origins, the two systems generated similar results as in singleplex qPCR. The duplex qPCR system, along with the sample preparation and DNA extraction specified in this study, constituted a first-reported duplex qPCR protocol for detection and quantification of the two blackleg pathogens from field samples.

Viral cross-class transmission results in disease of a phytopathogenic fungus.

Interspecies transmission of viruses is a well-known phenomenon in animals and plants whether via contacts or vectors. In fungi, interspecies transmission between distantly related fungi is often suspected but rarely experimentally documented and may have practical implications. A newly described double-strand RNA (dsRNA) virus found asymptomatic in the phytopathogenic fungus Leptosphaeria biglobosa of cruciferous crops was successfully transmitted to an evolutionarily distant, broad-host range pathogen Botrytis cinerea. Leptosphaeria biglobosa botybirnavirus 1 (LbBV1) was characterized in L. biglobosa strain GZJS-19. Its infection in L. biglobosa was asymptomatic, as no significant differences in radial mycelial growth and pathogenicity were observed between LbBV1-infected and LbBV1-free strains. However, cross-species transmission of LbBV1 from L. biglobosa to infection in B. cinerea resulted in the hypovirulence of the recipient B. cinerea strain t-459-V. The cross-species transmission was succeeded only by inoculation of mixed spores of L. biglobosa and B. cinerea on PDA or on stems of oilseed rape with the efficiency of 4.6% and 18.8%, respectively. To investigate viral cross-species transmission between L. biglobosa and B. cinerea in nature, RNA sequencing was carried out on L. biglobosa and B. cinerea isolates obtained from Brassica samples co-infected by these two pathogens and showed that at least two mycoviruses were detected in both fungal groups. These results indicate that cross-species transmission of mycoviruses may occur frequently in nature and result in the phenotypical changes of newly invaded phytopathogenic fungi. This study also provides new insights for using asymptomatic mycoviruses as biocontrol agent.

Molecular Interactions Between Leptosphaeria maculans and Brassica Species.

Canola is an important oilseed crop, providing food, feed, and fuel around the world. However, blackleg disease, caused by the ascomycete Leptosphaeria maculans, causes significant yield losses annually. With the recent advances in genomic technologies, the understanding of the Brassica napus-L. maculans interaction has rapidly increased, with numerous Avr and R genes cloned, setting this system up as a model organism for studying plant-pathogen associations. Although the B. napus-L. maculans interaction follows Flor's gene-for-gene hypothesis for qualitative resistance, it also puts some unique spins on the interaction. This review discusses the current status of the host-pathogen interaction and highlights some of the future gaps that need addressing moving forward.

Leptosphaeria maculans-Brassica napus Battle: A Comparison of Incompatible vs. Compatible Interactions Using Dual RNASeq.

Leptosphaeria maculans causes blackleg disease, which is one of the most destructive diseases of canola (Brassica napus L.). Due to the erosion of the current resistance in B. napus, it is pivotal to introduce new resistant genotypes to the growers. This study evaluated the potential of Rlm7 gene as resistance to its corresponding avirulence AvrLm7 gene is abundant. The Rlm7 line was inoculated with L. maculans isolate with AvrLm7; UMAvr7; and the CRISPR/Cas9 knockout AvrLm7 mutant, umavr7, of the same isolate to cause incompatible and compatible interactions, respectively. Dual RNA-seq showed differential gene expressions in both interactions. High expressions of virulence-related pathogen genes-CAZymes, merops, and effector proteins after 7-dpi in compatible interactions but not in incompatible interaction-confirmed that the pathogen was actively virulent only in compatible interactions. Salicyclic and jasmonic acid biosynthesis and signaling-related genes, defense-related PR1 gene (GSBRNA2T00150001001), and GSBRNA2T00068522001 in the NLR gene family were upregulated starting as early as 1- and 3-dpi in the incompatible interaction and the high upregulation of those genes after 7-dpi in compatible interactions confirmed the early recognition of the pathogen by the host and control it by early activation of host defense mechanisms in the incompatible interaction.

Two independent approaches converge to the cloning of a new Leptosphaeria maculans avirulence effector gene, AvrLmS-Lep2.

Brassica napus (oilseed rape, canola) seedling resistance to Leptosphaeria maculans, the causal agent of blackleg (stem canker) disease, follows a gene-for-gene relationship. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in B. napus 'Surpass 400'. Here we report cloning of AvrLmS and AvrLep2 using two independent methods. AvrLmS was cloned using combined in vitro crossing between avirulent and virulent isolates with sequencing of DNA bulks from avirulent or virulent progeny (bulked segregant sequencing). AvrLep2 was cloned using a biparental cross of avirulent and virulent L. maculans isolates and a classical map-based cloning approach. Taking these two approaches independently, we found that AvrLmS and AvrLep2 are the same gene. Complementation of virulent isolates with this gene confirmed its role in inducing resistance on Surpass 400, Topas-LepR2, and an RlmS-line. The gene, renamed AvrLmS-Lep2, encodes a small cysteine-rich protein of unknown function with an N-terminal secretory signal peptide, which is a common feature of the majority of effectors from extracellular fungal plant pathogens. The AvrLmS-Lep2/LepR2 interaction phenotype was found to vary from a typical hypersensitive response through intermediate resistance sometimes towards susceptibility, depending on the inoculation conditions. AvrLmS-Lep2 was nevertheless sufficient to significantly slow the systemic growth of the pathogen and reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field.

Genetic diversity and population structure of Leptosphaeria maculans isolates in Western Canada.

Leptosphaeria maculans is a serious concern for canola production worldwide. For effective disease management, knowledge of the pathogen's genetic variability and population structure is a prerequisite. In this study, whole-genome sequencing was performed for 162 of 1590 L. maculans isolates collected in the years 2007-2008 and 2012-2014 in Western Canada. DNA variants in genome-wide and specific regions including avirulence (Avr) genes were characterized. A total of 31,870 high-quality polymorphic DNA variants were used to study L. maculans genetic diversity and population structure. Cluster analysis showed that 150 isolates were clustered into 2 main groups and 4 subgroups by DNA variants located in either Avr or small secreted protein-encoding genes and into 2 main groups and 6 subgroups by genome-wide variants. The analysis of nucleotide diversity and differentiation also confirmed genetic variation within a population and among populations. Principal component analysis with genome-wide variants showed that the isolates collected in 2012-2014 were more genetically diverse than those collected in 2007-2008. Population structure analysis discovered three distinct sub-populations. Although isolates from Saskatchewan and Alberta were of similar genetic composition, Manitoba isolates were highly diverse. Genome-wide association study detected DNA variants in genes AvrLm4-7, Lema_T86300, and Lema_T86310 associated with the years of collection.

A new avirulence gene of Leptosphaeria maculans, AvrLm14, identifies a resistance source in American broccoli (Brassica oleracea) genotypes.

In many cultivated crops, sources of resistance to diseases are sparse and rely on introgression from wild relatives. Agricultural crops often are allopolyploids resulting from interspecific crosses between related species, which are sources of diversity for resistance genes. This is the case for Brassica napus (oilseed rape, canola), an interspecific hybrid between Brassica rapa (turnip) and Brassica oleracea (cabbage). B. napus has a narrow genetic basis and few effective resistance genes against stem canker (blackleg) disease, caused by the fungus Leptosphaeria maculans, are currently available. B. rapa diversity has proven to be a valuable source of resistance (Rlm, LepR) genes, while B. oleracea genotypes were mostly considered susceptible. Here we identified a new resistance source in B. oleracea genotypes from America, potentially effective against French L. maculans isolates under both controlled and field conditions. Genetic analysis of fungal avirulence and subsequent cloning and validation identified a new avirulence gene termed AvrLm14 and suggested a typical gene-for-gene interaction between AvrLm14 and the postulated Rlm14 gene. AvrLm14 shares all the usual characteristics of L. maculans avirulence genes: it is hosted in a genomic region enriched in transposable elements and heterochromatin marks H3K9me3, its expression is repressed during vegetative growth but shows a strong overexpression 5-9 days following cotyledon infection, and it encodes a small secreted protein enriched in cysteine residues with few matches in databases. Similar to the previously cloned AvrLm10-A, AvrLm14 contributes to reduce lesion size on susceptible cotyledons, pointing to a complex interplay between effectors promoting or reducing lesion development.

Analysis of the Oxidative Burst and Its Relevant Signaling Pathways in Leptosphaeria maculans-Brassica napus Pathosystem.

An oxidative burst is an early response of plants to various biotic/abiotic stresses. In plant-microbe interactions, the plant body can induce oxidative burst to activate various defense mechanisms to combat phytopathogens. A localized oxidative burst is also one of the typical behaviors during hypersensitive response (HR) caused by gene-for-gene interaction. In this study, the occurrence of oxidative burst and its signaling pathways was studied from different levels of disease severity (i.e., susceptible, intermediate, and resistant) in the B. napus-L. maculans pathosystem. Canola cotyledons with distinct levels of resistance exhibited differential regulation of the genes involved in reactive oxygen species (ROS) accumulation and responses. Histochemical assays were carried out to understand the patterns of H2O2 accumulation and cell death. Intermediate and resistant genotypes exhibited earlier accumulation of H2O2 and emergence of cell death around the inoculation origins. The observations also suggested that the cotyledons with stronger resistance were able to form a protective region of intensive oxidative bursts between the areas with and without hyphal intrusions to block further fungal advancement to the uninfected regions. The qPCR analysis suggested that different onset patterns of some marker genes in ROS accumulation/programmed cell death (PCD) such as RBOHD, MPK3 were associated with distinct levels of resistance from B. napus cultivars against L. maculans. The observations and datasets from this article indicated the distinct differences in ROS-related cellular behaviors and signaling between compatible and incompatible interactions.

Status and advances in mining for blackleg (Leptosphaeria maculans) quantitative resistance (QR) in oilseed rape (Brassica napus).

KEY MESSAGE: Quantitative resistance (QR) loci discovered through genetic and genomic analyses are abundant in the Brassica napus genome, providing an opportunity for their utilization in enhancing blackleg resistance. Quantitative resistance (QR) has long been utilized to manage blackleg in Brassica napus (canola, oilseed rape), even before major resistance genes (R-genes) were extensively explored in breeding programmes. In contrast to R-gene-mediated qualitative resistance, QR reduces blackleg symptoms rather than completely eliminating the disease. As a polygenic trait, QR is controlled by numerous genes with modest effects, which exerts less pressure on the pathogen to evolve; hence, its effectiveness is more durable compared to R-gene-mediated resistance. Furthermore, combining QR with major R-genes has been shown to enhance resistance against diseases in important crops, including oilseed rape. For these reasons, there has been a renewed interest among breeders in utilizing QR in crop improvement. However, the mechanisms governing QR are largely unknown, limiting its deployment. Advances in genomics are facilitating the dissection of the genetic and molecular underpinnings of QR, resulting in the discovery of several loci and genes that can be potentially deployed to enhance blackleg resistance. Here, we summarize the efforts undertaken to identify blackleg QR loci in oilseed rape using linkage and association analysis. We update the knowledge on the possible mechanisms governing QR and the advances in searching for the underlying genes. Lastly, we lay out strategies to accelerate the genetic improvement of blackleg QR in oilseed rape using improved phenotyping approaches and genomic prediction tools.

A conserved Zn2Cys6 transcription factor, identified in a spontaneous mutant from in vitro passaging, is involved in pathogenicity of the blackleg fungus Leptosphaeria maculans.

Continuous passaging in vitro can lead to the accumulation of changes in DNA sequence that potentially affect the properties of microbes, making them different from the original isolates. The identification of such genetic alterations is rare in fungi. A set of insertional mutants in the plant pathogenic fungus Leptosphaeria maculans, all derived from the same transformation experiment, had independent Agrobacterium T-DNA insertions and reduced pathogenicity on canola (Brassica napus). None of the insertions co-segregated in progeny from crosses with the reduction in pathogenicity. Genome sequences of three strains were analysed, and a mutation identified in a gene (ptf1, for pathogenicity-associated transcription factor 1) encoding a putative Zn2(II)Cys6 transcription factor. Homologs are found in other ascomycetes, and are required for pathogenicity by Fusarium graminearum, Fusarium oxysporum and Magnaporthe oryzae. The mutation in the L. maculans ptf1 gene co-segregates in progeny from crosses with the reduction in pathogenicity, a strain with an independent mutant allele isolated using CRISPR-Cas9 editing has reduced pathogenicity, and addition of wild type copies of the gene restores pathogenicity. Thus, this work defines a base pair substitution that occurred during in vitro passaging of a fungus that contributed to an attenuation of pathogenicity.

Constitutive expression of transcription factor SirZ blocks pathogenicity in Leptosphaeria maculans independently of sirodesmin production.

Sirodesmin, the major secondary metabolite produced by the plant pathogenic fungus Leptosphaeria maculans in vitro, has been linked to disease on Brassica species since the 1970s, and yet its role has remained ambiguous. Re-examination of gene expression data revealed that all previously described genes and two newly identified genes within the sir gene cluster in the genome are down-regulated during the crucial early establishment stages of blackleg disease on Brassica napus. To test if this is a strategy employed by the fungus to avoid damage to and then detection by the host plant during the L. maculans asymptomatic biotrophic phase, sirodesmin was produced constitutively by overexpressing the sirZ gene encoding the transcription factor that coordinates the regulation of the other genes in the sir cluster. The sirZ over-expression strains had a major reduction in pathogenicity. Mutation of the over-expression construct restored pathogenicity. However, mutation of two genes, sirP and sirG, required for specific steps in the sirodesmin biosynthesis pathway, in the sirZ over-expression background resulted in strains that were unable to synthesize sirodesmin, yet were still non-pathogenic. Elucidating the basis for this pathogenicity defect or finding ways to overexpress sirZ during disease may provide new strategies for the control of blackleg disease.

Hormonal Responses to Susceptible, Intermediate, and Resistant Interactions in the Brassica napus-Leptosphaeria maculans Pathosystem.

Hormone signaling plays a pivotal role in plant-microbe interactions. There are three major phytohormones in plant defense: salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). The activation and trade-off of signaling between these three hormones likely determines the strength of plant defense in response to pathogens. Here, we describe the allocation of hormonal signaling in Brassica napus against the fungal pathogen Leptosphaeria maculans. Three B. napus genotypes (Westar, Surpass400, and 01-23-2-1) were inoculated with two L. maculans isolates (H75 8-1 and H77 7-2), subsequently exhibiting three levels of resistance: susceptible, intermediate, and resistant. Quantitative analyses suggest that the early activation of some SA-responsive genes, including WRKY70 and NPR1, contribute to an effective defense against L. maculans. The co-expression among factors responding to SA/ET/JA was also observed in the late stage of infection. The results of conjugated SA measurement also support that early SA activation plays a crucial role in durable resistance. Our results demonstrate the relationship between the onset patterns of certain hormone regulators and the effectiveness of the defense of B. napus against L. maculans.

Genome-wide mapping of histone modifications during axenic growth in two species of Leptosphaeria maculans showing contrasting genomic organization.

Leptosphaeria maculans 'brassicae' (Lmb) and Leptosphaeria maculans 'lepidii' (Lml) are closely related phytopathogenic species that exhibit a large macrosynteny but contrasting genome structure. Lmb has more than 30% of repeats clustered in large repeat-rich regions, while the Lml genome has only a small amount of evenly distributed repeats. Repeat-rich regions of Lmb are enriched in effector genes, expressed during plant infection. The distinct genome structures of Lmb and Lml provide an excellent model for comparing the organization of pathogenicity genes in relation to the chromatin landscape in two closely related phytopathogenic fungi. Here, we performed chromatin immunoprecipitation (ChIP) during axenic culture, targeting histone modifications typical for heterochromatin or euchromatin, combined with transcriptomic analysis to analyze the influence of chromatin organization on gene expression. In both species, we found that facultative heterochromatin is enriched with genes lacking functional annotation, including numerous effector and species-specific genes. Notably, orthologous genes located in H3K27me3 domains are enriched with effector genes. Compared to other fungal species, including Lml, Lmb is distinct in having large H3K9me3 domains associated with repeat-rich regions that contain numerous species-specific effector genes. Discovery of these two distinctive heterochromatin landscapes now raises questions about their involvement in the regulation of pathogenicity, the dynamics of these domains during plant infection and the selective advantage to the fungus to host effector genes in H3K9me3 or H3K27me3 domains.