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Analyzing the function of TUP1 in rust fungi could provide a better understand of how it acts within the unique biological context in which these pathogens develop. Cys3 controls the synthesis of a set of catabolic enzymes for utilization of secondary sulfur sources when sulfur-containing amino acids and inorganic sulfate are missing or limited . The presence of a similar gene in Pst might suggest that secondary sulfur sources play a role in biotrophy, and upregulation of this gene in haustoria could suggest these sources are derived from the host. Cellular differentiation and filamentous growth are finely regulated in many fungi in response to environmental and nutritional signals.
External stimuli must be transmitted within the fungus by signal transduction pathways to enable the appropriate response. Our transcriptomic analysis revealed high expression of signal transduction components in both germinated spores and haustoria. Transcripts encoding proteins related to two component response regulators, the Ras protein superfamily of guanine nucleotide exchange factors, and diverse protein kinases were most highly represented. In fungi, two-component signalling systems participate in processes such as environmental sensing, oxidative stress response, cell-cycle control, and switching between non-pathogenic and pathogenic states  ,  , .
They are typically composed of a sensor-kinase protein, a phosphorelay protein, and a response regulator. We found genes encoding two-component regulatory proteins in both haustoria and germinated spores Table S6 , HeatMap. Generally, the proteins expressed in each tissue were distinct, consistent with the idea that they respond to different stimuli.
There was a larger representation of these genes in germinated spores suggesting a more complex interpretation of environmental signals prior to development of the haustorial structure. Os-1, also known as Nik-1, is required for adaptation to high osmolarity  , .
Three groups of selective fungicides target the osmotic stress signal transduction pathway  , . Intracellular osmotic pressure plays important roles in fungal development, for example as inside the M. Although disruption of HIK1 Os-1 in this pathogen caused no defect in growth on normal media or in pathogenicity to rice plants, the mutant strain acquired resistance to three groups of fungicides phenylpyrroles, dicarboximides, and aromatic hydrocarbons .
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The almost exclusive expression of this gene in Pst germinated spores suggests that the osmosensitive signal transduction pathway could play a role in the early stages of stripe rust disease. Ras proteins are a superfamily of guanine nucleotide exchange factors in which the phosphorylation status of the bound nucleotide operates a binary switch. They have numerous signaling roles in cellular functions including cytoskeletal integrity, proliferation, differentiation, cell adhesion, and cell migration.
We identified evidence of expression of genes encoding proteins with similarity to Ras proteins called ADP ribosylation factors ARF in both sampled tissues, which is of particular interest to rust pathogenesis because they regulate vesicular traffic, phospholipid metabolism and actin remodelling. Other genes encoding other types of Ras protein as well as GEFs and GAPs were also found in both tissues, suggesting complete representation of these complex molecular switches that regulate cell fate.
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Transcriptome studies such as the current work considerably enhance analysis of genome information. The platform used here gives significantly longer reads than Illumina, which both helps with transcript assembly and demonstrates gene expression. In contrast, small-read based assemblies are subject to assembly errors and the uncertainties of gene prediction, although these also benefit from annotation with transcriptomic data.
One important aspect of defining transcriptomes, as described here, is that it cuts through the complexity of the genome. Presumably, many of the remaining genes are expressed in different contexts for example on the alternate host, or as part of different spore stages , or were simply undetected or unassembled in our study.
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Although isolation of haustoria is a very powerful way to track the pathogenic stage of the fungus, we did not analyse infected tissue which includes the infectious hyphal stage that presumably also secretes effector proteins. Another important aspect of our study is quantification of expression using Illumina data, which considerably enhances the accuracy and reliability of our expression measurements. Thus, use of and Illumina together provides a robust expression data set. This allowed us to predict a suite of potential effector genes, defined chiefly as genes upregulated in haustoria that encode secreted proteins.
We also note that not all secreted proteins will be virulence effectors, and some within our dataset show clear hallmarks of roles in fungal cell wall modification. The current data provide a comprehensive view of the stripe rust transcriptome in germinated spores and haustorial stages. Overall, the fungus seems to use similar biochemical pathways to those described in better characterised species such as M. Haustoria and spores are clearly working in different ways. The germinating spores prioritises polarised growth from stored energy until such time as it is able to mine its own resources, whereas haustoria are a sessile stage devoted to nutrient extraction and defence suppression.
The uredinospore contains vast stores of energy in the form of lipid bodies, sugar alcohols and proteins  ,  to allow the foraging germling to find the pathogenic niche, and in that sense is analogous to a plant seed. From our data, we can see that haustoria create abundant demand for sugars, amino acids, and nitrogen, through high expression of transporters for these fundamental building blocks. In addition, we propose a new mechanism for sulphur assimilation by uptake of methylmethionine.
Sugars are used immediately by haustoria for the production of ATP through glycolysis, TCA cycle and oxidative phosphoryation. By contrast, spores seem to obtain energy by utilising lipid resources via the glyoxylate pathway. A further important haustorial function is biosynthesis of macromolecules, and it is clear that steady production of the reductant NADPH is made possible by import of nicotinic acid through dedicated transporters, and its ultimate conversion to NADPH by the PPS.
Spores also use the PPS but it is likely that this is works in the opposite oxidative direction, for energy production. Molecules made in haustoria are presumably exported directly to spores for biogenesis and storage, so the molecular pathways active in haustoria should reflect spore composition directly. With respect to the completeness of our dataset, we note as have previous authors that many genes are novel so cannot be annotated.
For this reason, it is difficult to exclude the presence of certain genes with major implications for the biotrophic lifecycle, such as N and S assimilation. Of course, the absence of these genes could also be ascribed to low expression of miss-assembly of small reads. These are important challenges for the field because barring technological breakthroughs, most fungal genome sequences are likely to have large components of small sequence reads in their genesis.
Seven-day old seedlings were inoculated with fresh uredinospores of P. About 20 grams of heavily infected tissue were collected 9 days after infection dai; just prior to sporulation and immediately processed for haustoria isolation.
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For based transcriptome analysis, haustoria were isolated from stripe rust-infected Morocco leaves at 9 dai by Concanavalin A affinity chromatography Text S1 and Figure S4 . Half a plate was used following standard procedures recommended by Roche. Initial quality filtering of the raw reads was performed using Roche proprietary analysis software Newbler software release 2. Libraries from haustoria samples and germinated spores samples were sequenced with base paired-end reads.
CLC Genomic Workbench 4. The parameters used for de novo assembly were similarity 0. The mapping of Illumina reads against reference contigs was done after removing adapter sequences and reads of low quality trim quality score 0. Reads that mapped to multiple sites were assigned randomly and proportionally to one of the mapped sites. The random distribution was done proportionally to the number of unique matches that the genes to which it matches have, normalized by the transcript length. Three biological replicates of each tissue were used for differential gene expression analysis.
The genes with a false discovery rate FDR corrected P -value less than 0. Only the transcripts that belonged to one of the functional categories were analysed manually to verify if more than one transcript represented the same gene, in which case it was annotated in Tables S3 — S6. For the remainder of the transcripts, which comprise anonymous sequences, this was not possible because different transcripts might have the same top hit without meaning that they were derived from the same gene.
The haustorial transcriptome was further analysed with SignalP 3. Number of reads per assembled contig for germinated spores and haustorial samples. Length of contigs for germinated spores and haustorial samples assembled from data. Coverage of contigs assembled from data from germinated spores and haustoria samples.
Isolated haustoria by affinity chromatography. Stripe rust haustoria isolated by affinity chromatography. Bright field image with differential-interference contrast, haustoria black arrows and contaminating chloroplasts can be seen. Fluorescence microscopy of isolated haustoria by affinity chromatography.
Haustoria were isolated from infected tissue by ConA affinity chromatography as described in Materials and Methods. All images were collected on a Leica DMR epifluorescence microscope. Transcripts reference set. Haustorial secreted proteins HSPs.