Core regulated transcripts of V. longisporum in in situ (xylem-sap) and in

The first part of the chapter will be focused on the group of same-regulated transcripts of V. longisporum 43 during in situ and in vitro cultivation. 38% of all transcripts are genes with unchanged abundance. These 8,121 genes were expressed but not regulated with regard to cultivation in xylem-sap and SXM and are the core set of expressed transcripts. In Figure 27, the 1-D self organizing map visualizes the clustering of all transcripts. The analyzed similary regulated 8,121 core-regulated transcripts were marked with a red rectangle.

Figure 27: Verticillium longisporum 43 transcripts up-regulated in xylem-sap and in SXM. MarVis cluster (1-D self organiszing map) of all 21,192 transcripts that are regulated in SXM and xylem-sap. Up-regulated transcripts are shown in red (1) left: SXM, right: xylem-sap. Not regulated tanscripts are shown in blue (0), left:

xylem-sap, right: SXM. Transcripts that are up-regulated under both conditions and being described in this chapter, are marked with a red rectangle (http://marvis.gobics.de, (Kaever et al., 2009)).

For these core-regulated transcripts the cellular localization, functional categorization and domain function of the transcripts encoded proteins will be analyzed. Exel sheets of the data were attached on CD.

3.3.1.1 Cellular localization of core transcripts

To analyze the putative localization of those transcripts, the prediction tools WoLF PSORT (Horton et al., 2007) and YLoc+ (Briesemeister et al., 2010) were used (Figure 28).

Figure 28: Cellular localization of core set transcripts predicted by WoLF PSORT and YLoc+.

(a) WoLF PSORT (Horton et al., 2007) analysis reveals most of the predicted proteins to be localized in the nucleus. In the mitochondria, plasma membrane, cytoplasm and extracellular also many proteins seem to be localized. (b) YLoc+ (Briesemeister et al., 2010) analysis reveals about half of the proteins to be in the cytoplasma.

About one quarter is nuclear localized and less were plasma membrane, mitochondrial or secreted proteins.

The prediction results of the two programmes differ. YLoc+ assigned the cellular localization for much more transcripts then WoLF PSORT. Most of the 6,427 YLoc+ predicted proteins seem to be localized in the cytoplasm (3,324) or are nuclear proteins (1,627). In total 80% of the core regulated transcripts were assigned for localization by this tool. WoLF PSORT predicted a locally determination for 4,859 transcripts what is about 50% in total. Nuclear proteins are with 1,611 the biggest group. For cytoplama localization only 684 proteins were predicted what is a fractional amount of the YLoc+ assignment. Finally, both predictions revealed the core set of regulated transcripts to encode for proteins being localized nuclear, in the cytoplasm and at or in the plasma membrane.

3.3.1.2 Comparison of functional classes and predicted domains of the core transcriptome

To get more information of putative functions of the core-regulated transcripts encoded proteins either in xylem-sap or in SXM, the transcripts were sorted to functional categories using FunCat (http://mips.helmholtz-muenchen.de/proj/funcatDB/) and their domains were predicted usind InterproScan. Filtering and sorting for functional categorization was performed by using the “Hivi” tool (www.gobics.de) with a cutoff of 50% protein sequence identity to ensure a reasonable sorting into the functional groups. Proteins were predicted by comparison with the soil-borne plant pathogenic fungus Fusarium graminearum.

The predicted proteins were split up in 19 functional main groups. In these groups, 7,581 transcript encoded proteins were ordered to, what is about 93% of the same regulated transcripts.

Most of the putative predicted proteins seem to be “metabolism” involved, where transcripts for synthesis as well as for degradation were assigned to. Putative proteins being involved in

“cellular transport”, “protein with binding function or co-factor requirement”, “protein fate” and

“transcription” were also detected in high numbers. Proteins with these functions are required for fungal growth in situ (xylem-sap) and in vitro (SXM) and seem to belong to the core set of proteins in Verticillium longisporum.

The main groups and the first 30 functional subcategories for the encoded proteins are analyzed (Table 11). By this it is possible to get a more detailed view of the 7,581 predicted functions for the core regulated transcripts in the in situ and the in vitro medium. In the table, main categories are written in italic and the subcategories not. For analyzing these data it has to be mentioned, that a putative protein could be sorted to more than one functional group. This is caused by the fact, that the proteins might have several functions.

Table 11: First 30 functional subcategories for xylem-sap (xyS) and SXM same regulated transcripts

Category with subcategories / genes in this category xyS and SXM up

lipid, fatty acid and isoprenoid metabolism 307

metabolism of vitamins, co-factors, and prosthetic groups 136

secondary metabolism 190

Protein with binding function or co-factor requirement 843 (structural or catalytic)

cellular sensing and response to external stimulus 159

Energy 272

Table 11 continued

Category with subcategories / genes in this category xyS and SXM up

Regulation of metabolism and protein function 157

regulation of protein activity 139

Development (systemic) 39

Systemic interaction with the environment 10 Transposable elements, viral and plasmid proteins 3

Organ differentiation 2

7581

As expected, the core transcription reveals all essential functions of living cells. The four subgroups of “C-compound and carbohydrate metabolism”, “RNA synthesis”,

“transported compounds (substrates)” and “transport routes” are with over 400 predicted proteins the fourth highest up-regulated subcategories of core expressed transcripts in the xylem-sap and the pectin rich SXM medium. With a number of 534 (14% of the over 7,500 assigned proteins of core regulated transcripts), most encoded proteins are involved in

“C-compound and carbohydrate metabolism” and many of them are putative CAZys (carbohydrate-active enzymes) that are also predicted by direct Pfam domain analysis of the dataset. The high number of core regulated putative CAZy proteins highlighted the importance of those enzymes for the fungus. Furthermore, transcripts coding for “RNA synthesis” proteins (main group of “transcription”), like the probable RPO31- DNA-directed RNA polymerase III, 160 KD subunit, being encoded by vl43-au16.g10116.t1 are highly up-regulated. Pfam analysis assisted the importance of those proteins. Here, especially ribosomal domains were detected in highest numbers. Also transcription factors were found by Pfam analysis to be represented in high amounts. The CPC1 genes (vl43-au16.g19783.t1 and vl43-au16.g20638.t1) of the transcription factor, which is the activator of the cross-pathway control system of amino acid biosynthesis, and is relevant for Verticillium pathogenicity (Sasse et al., 2008; Timpner et al., 2013), were found to be core regulated. The genes were both found to be expressed in a higher level over 130 RPKM but could not be assigned to a functional group.

Transcription factors were found as well in the subgroup “nucleic acid binding” of the main category of “protein with binding functions”. For these binding proteins also the Pfam analysis detects lots of domains what is also the case for transporter domains especially sugar transporters that are needed by the fungus. Transport plays also an important role for Verticillium longisporum 43. In “transported compounds (substrates)” and “transport routes”.

Transcripts of this functional group were found in high numbers to be core-regulated. Summing up, beside proteins with functions as ribosomal proteins, CAZys, transcription factors, binding proteins and transport proteins, the Pfam analysis and the analysis by categorization revealed lots of enzymes especially peptidases and kinases. That emphasized the importance of these proteins for the fungus.

Beside CPC1, the pathogenicity relevant PA14_2 gene (vl43-au16.g5346.t1), which is characterized in this study, was found to be core-regulated. With RPKM values around 2 it was found to be less expressed. Unfortionately also the PA14_2 gene could not be assigned to a functional category by the F. graminearum comparison using the “Hivi” tool.

3.3.2 Specific regulated transcripts of V. longisporum in in situ (xylem-sap) and in vitro