The basidiomycete Melampsora larici-populina causes poplar rust disease by invading leaf tissues and secreting effector proteins through specialized infection structures known as haustoria. The mechanisms by which rust effectors promote pathogen virulence are poorly understood. The present study characterized Mlp124478, a candidate effector of M. larici-populina. We used the models Arabidopsis thaliana and Nicotiana benthamiana to investigate the function of Mlp124478 in plant cells. We established that Mlp124478 accumulates in the nucleus and nucleolus, however its nucleolar accumulation is not required to promote growth of the oomycete pathogen Hyaloperonospora arabidopsidis. Stable constitutive expression of Mlp124478 in A. thaliana repressed the expression of genes involved in immune responses, and also altered leaf morphology by increasing the waviness of rosette leaves. Chip-PCR experiments showed that Mlp124478 associats'e with the TGA1a-binding DNA sequence. Our results suggest that Mlp124478 exerts a virulence activity and binds the TGA1a promoter to suppress genes induced in response to pathogen infection.

Background: RNA sequencing allows the measuring of gene expression at a resolution unmet by expression arrays or RT-qPCR. It is however necessary to normalize sequencing data by library size, transcript size and composition, among other factors, before comparing expression levels. The use of internal control genes or spike-ins is advocated in the literature for scaling read counts, but the methods for choosing reference genes are mostly targeted at RT-qPCR studies and require a set of pre-selected candidate controls or pre-selected target genes. Results: Here, we report an R-based pipeline to select internal control genes based solely on read counts and gene sizes. This novel method first normalizes the read counts to Transcripts per Million (TPM) and then excludes weakly expressed genes using the DAFS script to calculate the cut-off. It then selects as references the genes with lowest TPM coefficient of variation. We used this method to pick custom reference genes for the differential expression analysis of three transcriptome sets from transgenic Arabidopsis plants expressing heterologous fungal effector proteins tagged with GFP (using GFP alone as the control). The custom reference genes showed lower coefficient of variation and fold change as well as a broader range of expression levels than commonly used reference genes. When analyzed with NormFinder, both typical and custom reference genes were considered suitable internal controls, but the custom selected genes were more stably expressed. geNorm produced a similar result in which most custom selected genes ranked higher (i.e. were more stably expressed) than commonly used reference genes. Conclusions: The proposed method is innovative, rapid and simple. Since it does not depend on genome annotation, it can be used with any organism, and does not require pre-selected reference candidates or target genes that are not always available.

Chaga (Inonotus obliquus) is a medicinal fungus used in traditional medicine of Native American and North Eurasian cultures. Several studies have demonstrated the medicinal properties of chaga’s bioactive molecules. For example, several terpenoids (e.g., betulin, betulinic acid and inotodiol) isolated from I. obliquus cells have proven effectiveness in treating different types of tumor cells. However, the molecular mechanisms and regulation underlying the biosynthesis of chaga terpenoids remain unknown. In this study, we report on the optimization of growing conditions for cultured I. obliquus in presence of different betulin sources (e.g., betulin or white birch bark). It was found that better results were obtained for a liquid culture pH 6.2 at 28 °C. In addition, a de novo assembly and characterization of I. obliquus transcriptome in these growth conditions using Illumina technology was performed. A total of 219,288,500 clean reads were generated, allowing for the identification of 20,072 transcripts of I. obliquus including transcripts involved in terpenoid biosynthesis. The differential expression of these genes was confirmed by quantitative-PCR. This study provides new insights on the molecular mechanisms and regulation of I. obliquus terpenoid production. It also contributes useful molecular resources for gene prediction or the development of biotechnologies for the alternative production of terpenoids.

Melampsora larici-populina (Mlp) is a devastating pathogen of poplar trees, causing the defoliating poplar leaf rust disease. Genomic studies have revealed that Mlp possesses a repertoire of 1184 small secreted proteins (SSPs), some of them being characterized as candidate effectors. However, how they promote virulence is still unclear. This study investigates the candidate effector Mlp37347’s role during infection. We developed a stable Arabidopsis transgenic line expressing Mlp37347 tagged with the green fluorescent protein (GFP). We found that the effector accumulated exclusively at plasmodesmata (PD). Moreover, the presence of the effector at plasmodesmata favors enhanced plasmodesmatal flux and reduced callose deposition. Transcriptome profiling and a gene ontology (GO) analysis of transgenic Arabidopsis plants expressing the effector revealed that the genes involved in glucan catabolic processes are up-regulated. This effector has previously been shown to interact with glutamate decarboxylase 1 (GAD1), and in silico docking analysis supported the strong binding between Mlp37347 and GAD1 in this study. In infection assays, the effector promoted Hyalonoperospora arabidopsidis growth but not bacterial growth. Our investigation suggests that the effector Mlp37347 targets PD in host cells and promotes parasitic growth.

There is increasing evidence that mobile genetic elements can drive the emergence of pathogenic fungal species by moving virulence genes horizontally. The 14 kbp ToxhAT transposon has been shown to be moving the necrotrophic effector, ToxA, horizontally between fungal species that infect Triticum aestivum (wheat), namely Parastagonospora nodorum , Pyrenophora tritici-repentis , and Bipolaris sorokiniana . All three species utilise the ToxA protein to infect wheat. Previous genomic evidence found ToxhAT in distinct chromosomal positions in two isolates of B. sorokiniana , indicating that the transposon is still active in this species. Here we confirm the movement of ToxhAT using long-read Nanopore MinION sequencing of eight novel and one previously published B. sorokiniana isolates. One event of independent transposition of ToxhAT was observed, and target site duplications of “TA” were identified, confirming this was an autonomous movement facilitated by a yet unidentified transposase. Whole genome analysis revealed that ToxhAT is a passenger embedded in a much larger, conserved 170–196 kbp mobile genetic element. This element, termed Sanctuary , belongs to the newly described Starship transposon superfamily. This classification is based on the presence of short direct repeats, empty insertion sites, a putative tyrosine recombinase gene and other features of Starship transposons. We also show that ToxhAT has been independently captured by two different Starships , Sanctuary and Horizon which share little to no sequenced identity, outside of ToxhAT. This classification makes Horizon and Sanctuary part of a growing number of Starships involved in the horizontal gene transfer of adaptive genetic material between fungal species. Importance: The work presented here expands our understanding of a novel group of mobile genetic elements called Starships that facilitate the horizontal exchange of virulence genes in fungal pathogens. Our analysis shows that Sanctuary and ToxhAT are likely active and autonomous transposons in the B. sorokiniana genome. We also show that the smaller ToxhAT transposon has been independently captured by two different Starships , viz. Sanctuary in B. sorokiniana and Horizon in P. tritici-repentis and P. nodorum. Outside of ToxhAT these two Starships share no sequence identity. The capture of ToxhAT by two different mobile elements in three different fungal wheat pathogens demonstrates how horizontal transposon transfer is driving the evolution of virulence in these important wheat pathogens.