Interaction of roses with a biotrophic and a hemibiotrophic leaf pathogen leads to differences in defense transcriptome activation.

KEY MESSAGE: Transcriptomic analysis resulted in the upregulation of the genes related to common defense mechanisms for black spot and the downregulation of the genes related to photosynthesis and cell wall modification for powdery mildew. Plant pathogenic fungi successfully colonize their hosts by manipulating the host defense mechanisms, which is accompanied by major transcriptome changes in the host. To characterize compatible plant pathogen interactions at early stages of infection by the obligate biotrophic fungus Podosphaera pannosa, which causes powdery mildew, and the hemibiotrophic fungus Diplocarpon rosae, which causes black spot, we analyzed changes in the leaf transcriptome after the inoculation of detached rose leaves with each pathogen. In addition, we analyzed differences in the transcriptomic changes inflicted by both pathogens as a first step to characterize specific infection strategies. Transcriptomic changes were analyzed using next-generation sequencing based on the massive analysis of cDNA ends approach, which was validated using high-throughput qPCR. We identified a large number of differentially regulated genes. A common set of the differentially regulated genes comprised of pathogenesis-related (PR) genes, such as of PR10 homologs, chitinases and defense-related transcription factors, such as various WRKY genes, indicating a conserved but insufficient PTI [pathogen associated molecular pattern (PAMP) triggered immunity] reaction. Surprisingly, most of the differentially regulated genes were specific to the interactions with either P. pannosa or D. rosae. Specific regulation in response to D. rosae was detected for genes from the phenylpropanoid and flavonoid pathways and for individual PR genes, such as paralogs of PR1 and PR5, and other factors of the salicylic acid signaling pathway. Differently, inoculation with P. pannosa leads in addition to the general pathogen response to a downregulation of genes related to photosynthesis and cell wall modification.

GWAS reveals a rapidly evolving candidate avirulence effector in the Cercospora leaf spot pathogen.

The major resistance gene BvCR4 recently bred into sugar beet hybrids provides a high level of resistance to Cercospora leaf spot caused by the fungal pathogen Cercospora beticola. The occurrence of pathogen strains that overcome BvCR4 was studied using field trials in Switzerland conducted under natural disease pressure. Virulence of a subset of these strains was evaluated in a field trial conducted under elevated artificial disease pressure. We created a new C. beticola reference genome and mapped whole genome sequences of 256 isolates collected in Switzerland and Germany. These were combined with virulence phenotypes to conduct three separate genome-wide association studies (GWAS) to identify candidate avirulence genes. We identified a locus associated with avirulence containing a putative avirulence effector gene named AvrCR4. All virulent isolates either lacked AvrCR4 or had nonsynonymous mutations within the gene. AvrCR4 was present in all 74 isolates from non-BvCR4 hybrids, whereas 33 of 89 isolates from BvCR4 hybrids carried a deletion. We also mapped genomic data from 190 publicly available US isolates to our new reference genome. The AvrCR4 deletion was found in only one of 95 unique isolates from non-BvCR4 hybrids in the United States. AvrCR4 presents a unique example of an avirulence effector in which virulent alleles have only recently emerged. Most likely these were selected out of standing genetic variation after deployment of BvCR4. Identification of AvrCR4 will enable real-time screening of C. beticola populations for the emergence and spread of virulent isolates.

P Starvation in Roses Leads to Strongly Genotype-Dependent Induction of P-Transporter Genes during Black Spot Leaf Disease.

Phosphorous starvation in plants has been reported to have contrasting effects on the interaction with pathogens in different plant pathogen systems and plant species. Both increases and decreases in susceptibility have been observed in numerous reports. Here, we analysed black spot infection and the leaf expression of two plant phosphate transporters and one defence marker gene in roses after phosphorous starvation. We varied three factors: phosphate starvation versus full supply of phosphorous, black spot infection vs. mock inoculation, and different susceptible and resistant progeny of a biparental rose population. Black spot susceptibility or resistance was not significantly changed upon phosphate starvation in either compatible or incompatible interactions. The expression of phosphate transporters was strongly induced upon starvation, but in some genotypes, expression was altered by black spot interaction as well. The marker for pathogenic interactions was exclusively induced by interaction with black spot, but the expression was altered by a combination of phosphate starvation and interaction with the fungus in some genotypes. In summary, phosphate starvation has clear effects on the gene expression of phosphate transporters in rose leaves, and the interaction with a hemibiotrophic leaf pathogen is strongly genotype dependent.

Prediction of the Diplocarpon rosae secretome reveals candidate genes for effectors and virulence factors.

Rose black spot is one of the most severe diseases of field-grown roses. Though R-genes have been characterised, little information is known about the molecular details of the interaction between pathogen and host. Based on the recently published genome sequence of the black spot fungus, we analysed gene models with various bioinformatic tools utilising the expression data of infected host tissues, which led to the prediction of 827 secreted proteins. A significant proportion of the predicted secretome comprises enzymes for the degradation of cell wall components, several of which were highly expressed during the first infection stages. As the secretome comprises major factors determining the ability of the fungus to colonise its host, we focused our further analyses on predicted effector candidates. In total, 52 sequences of 251 effector candidates matched several bioinformatic criteria of effectors, contained a Y/F/WxC motif, and did not match annotated proteins from other fungi. Additional sequences were identified based on their high expression levels during the penetration/haustorium formation phase and/or by matching known effectors from other fungi. Several host genotypes that are resistant to the sequenced isolate but differ in the R-genes responsible for this resistance are available. The combination of these genotypes with functional studies of the identified candidate effectors will allow the mechanisms of the rose black spot interaction to be dissected.

A draft genome sequence of the rose black spot fungus Diplocarpon rosae reveals a high degree of genome duplication.

BACKGROUND: Black spot is one of the most severe and damaging diseases of garden roses. We present the draft genome sequence of its causative agent Diplocarpon rosae as a working tool to generate molecular markers and to analyze functional and structural characteristics of this fungus. RESULTS: The isolate DortE4 was sequenced with 191x coverage of different read types which were assembled into 2457 scaffolds. By evidence supported genome annotation with the MAKER pipeline 14,004 gene models were predicted and transcriptomic data indicated that 88.5% of them are expressed during the early stages of infection. Analyses of k-mer distributions resulted in unexpectedly large genome size estimations between 72.5 and 91.4 Mb, which cannot be attributed to its repeat structure and content of transposable elements alone, factors explaining such differences in other fungal genomes. In contrast, different lines of evidences demonstrate that a huge proportion (approximately 80%) of genes are duplicated, which might indicate a whole genome duplication event. By PCR-RFLP analysis of six paralogous gene pairs of BUSCO orthologs, which are expected to be single copy genes, we could show experimentally that the duplication is not due to technical error and that not all isolates tested possess all of the paralogs. CONCLUSIONS: The presented genome sequence is still a fragmented draft but contains almost the complete gene space. Therefore, it provides a useful working tool to study the interaction of D. rosae with the host and the influence of a genome duplication outside of the model yeast in the background of a phytopathogen.