V. longisporum elicits media-dependent secretome responses with a further capacity to distinguish between plant-related environments

Verticillia cause a vascular wilt disease affecting a broad range of economically valuable crops. The fungus enters its host plants through the roots and colonizes the vascular system. It requires extracellular proteins for a successful plant colonization. The exoproteome of the allodiploid Verticillium longisporum was analyzed upon cultivation in different media. Secreted fungal proteins were identified by label free LC-MS/MS screening. V. longisporum induced two main secretion patterns. One response pattern was elicited in various non-plant related environments. The second pattern includes the exoprotein responses to the plant-related media, pectin-rich simulated xylem medium and pure xylem sap, which exhibited similar but additional distinct features. These exoproteomes include a shared core set of 223 secreted and similarly enriched fungal proteins. The pectin-rich medium significantly induced the secretion of 144 proteins including a number of pectin degrading enzymes, whereas xylem sap triggered a smaller but unique fungal exoproteome pattern with 32 enriched proteins. The latter pattern included proteins with domains of known effectors, metallopeptidases and carbohydrate-active enzymes. The most abundant and uniquely enriched proteins of these different groups are the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3, the cerato-platanin proteins Cp1 and Cp2, the metallopeptidases Mep1 and Mep2 and the CAZys Gla1, Amy1 and Cbd1. Deletion of the majority of the corresponding genes caused no phenotypic changes during ex planta growth or invasion and colonization of tomato plants. However, we discovered that the NLP2 and NLP3 deletion strains were compromised in plant infections. Overall, our exoproteome approach revealed that the fungus induces specific secretion responses in different environments. The fungus has a general response to non-plant related media whereas it is able to fine-tune its exoproteome in the presence of plant material. Importantly, the xylem sap-specific exoproteome pinpointed Nlp2 and Nlp3 as single effectors required for successful V. dahliae colonization. Author Summary Verticillium spp. infect hundreds of different plants world-wide leading to enormous economic losses. Verticillium wilt is a disease of the vasculature. The fungus colonizes the xylem of its host plant where it exploits the vascular system to colonize the whole plant. Therefore, the fungus spends part of its lifetime in this nutrient-low and imbalanced environment where it is inaccessible for disease control treatments. This lifestyle as well requires the fungus to react to plant defense responses by secreting specific effector molecules to establish a successful infection. We addressed the differences in media-dependent secretion responses of Verticillium longisporum. We identified a broad response pattern induced by several media, and a similar response (but with some distinct differences) for the plant-related environments: the pectin-rich medium SXM and xylem sap from the host rapeseed. Importantly, we show that the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3 are xylem sap-specific proteins that are required for full V. dahliae pathogenicity on tomato. These factors play a role during the colonization phase and represent potential targets for new control strategies for Verticillium wilt.

In this study, we extended the comparative analysis with rapeseed xylem sap and 144 focused on the fungal secretome. V. longisporum secreted proteins that were derived 145 from cultivation in different growth media were identified by a proteomic approach and 146 the protein patterns induced by different environments were compared. Our goal was 147 to obtain a more comprehensive overview of the secreted factors of V. longisporum in 148 response to different substances in its environment that putatively reflect different 149 stages of the infection. We analyzed the exoproteomes of V. longisporum on a broad 150 range of media from water to minimal and complete media. As an additional condition, 151 we applied simulated xylem medium (SXM), which is rich in pectin and which was 152 To obtain a more comprehensive analysis, complete lanes of the gels with the 188 respective exoproteomes were fractionally subjected to tryptic protein digestion and 189 the resulting peptides were analyzed by LC MS/MS . The obtained raw data were  190  channeled  through  a  bioinformatics  pipeline  based  on  Proteome  191 Discoverer Software 1.3™ (Thermo Scientific) and an in-house genome-wide protein 192 sequence database of V. longisporum. The received spectral counts were compared 193 on single secreted protein level by color-coded one-dimensional self-organizing maps 194 ( Fig 1B). These revealed that proteins that were strongly enriched in xylem sap or SXM 195 were not enriched in any other condition. Differences in the exoproteome signatures 196 are also illustrated by sample clustering in a principle component analysis plot (Fig  197   1C). Exoproteomes of V. longisporum derived from very diverse media including 198 nutrient-free water, V8 juice, CDM or YNB medium show a similar pattern. 199 Supplementation of CDM with B. napus plant proteins or xylem sap with a final 200 concentration of 7% did not result in a different exoproteome pattern, neither did 201 glucose supplementation to water. Therefore, the respective results for these 202 conditions were combined together. In contrast, proteins secreted in pectin-rich SXM 203 or xylem sap each showed a distinct pattern in comparison to the other media 204 conditions. These patterns representing the latter exoproteomes are similar in some 205 features as the clusters lie close to each other on the x-axis, although some differences 206 are present as analyzed further below. 207 Overall, our analysis illustrates that the fungus has the potential to form a general 208 secretome response to non-plant related environments and, in addition, a similar, but 209 more specialized response to plant-related substances (Fig 1C).  The proteins were precipitated from the culture  217 supernatants, subjected to LC MS/MS and analyzed. The data set was filtered with a 218 statistical workflow using MarVis-Suite [34]. S1 Table shows the identities of the 445  219 proteins from an in-house database with their protein sequences and details on their 220 abundance as measured by identified peptides. The list is sorted according to the most 221 specifically enriched proteins in xylem sap (green) and SXM (red), respectively. CAZy database (http://www.cazy.org) and dbCAN2 [37]. All details are given in S2 248 Table. The Venn diagram in Fig Table). 271 Further analysis of proteins from functional groups regarding the induction by different 272 media showed that pectin-rich medium predominantly triggered the secretion of 273 carbohydrate-degrading enzymes, but also peptidases and redox enzymes (Fig 2D,  274 S3 Table). No effectors were found in the SXM-specific exoproteome.  Table). In the proteins from the pectin-rich medium condition, the 285 CAZys are highly represented with 64 proteins whereas in xylem sap only 19 CAZys 286 are specifically enriched and 90 proteins belong to the core exoproteome (S4 Table). 287 The core exoproteome exhibits an overrepresentation of CAZy families with 32 288 proteins acting on pectin, including members of family GH28 (five proteins), PL1 (16 289 proteins), PL3 (seven proteins) and CE8 (four proteins). Additionally, the SXM-specific 290 and most enriched CAZy families comprise 20 pectin-degrading enzymes (families 291 GH28, PL1 and CE12 with ten, six and four proteins, respectively, S4 Table). Only a 292 few CAZys were specifically enriched in the xylem sap condition, and these were 293 distributed in different families. 294 Overall, we found that B. napus xylem sap and pectin-rich SXM, employed as plant-295 related culture environments, predominantly induced the secretion of carbohydrate-296 degrading enzymes. Compared to the rapeseed xylem sap condition, SXM triggered 297 an additional set of CAZys that were specifically enriched after cultivation in this 298 medium. 299 300

Xylem sap triggers the secretion of potential and known Verticillium effectors 301
Compared to the pectin-rich SXM, V. longisporum formed a more specific secretion 302 response in xylem sap with only 32 proteins that are uniquely enriched. This indicates 303 that the fungus can distinguish between xylem sap and the presence of other plant 304 material and accordingly fine-tunes its protein secretion. Furthermore, the proteins 305 secreted in the host xylem sap might be specifically important during plant colonization. 306 Table 1 displays the xylem sap-specific proteins. The corresponding isogenes are 307 paired up and the best hit in V. dahliae JR2 is given, except for the V. alfalfae specific 308 proteins that were searched against the V. alfalfae VaMs.102 proteome (Ensembl 309 Fungi). Within the identified groups, proteins were ranked according to the quotient of 310 peptide counts identified in xylem sap (XyS) by the number detected in the pectin-rich 311 SXM. Displayed peptide counts were averaged from 6 biological replicates. If the 312 number was below 1, it was calculated as 0 and the quotient was given as the average 313 XyS peptide counts (>). The 32 xylem sap-specific proteins comprise 15 'Proteins 314 acting on carbohydrates', five 'Proteins with domains interacting with carbohydrates', 315 five 'Effectors', four 'Peptidases', two 'Proteins involved in redox processes' and one 316 with a ubiquitin binding domain that was grouped as 'Other' (Table 1)  Chosen proteins are highlighted in Table 1  Additionally, the strains were tested for the involvement in stress responses with at 371 least one stressor tested for each strain. The stress inducing agent was added to 372 minimal medium. The cell wall perturbing agents SDS and ethanol or the oxidative 373 stressor hydrogen peroxide were used. All single deletion, double deletion and 374 complementation strains exhibited a similar morphological development to V. dahliae 375 WT, which is exemplified by growth on SXM (Fig 3). 376 Overall, these results suggest that Cp1, Cp2, Nlp2, Nlp3, Mep1, Mep2, Gla1, Cbd1 377 and Amy1, that were found to be enriched specifically in xylem sap cultures, are 378 dispensable for vegetative growth, development and stress response of V. dahliae. indistinguishable between WT/OE-GFP and ∆NLP3/OE-GFP (Fig 7A). Initial root 444 colonization was observed at three days following inoculation and whole roots were 445 covered with fungal hyphae after five days suggesting that Nlp3 is dispensable for 446

A. thaliana root colonization. 447
Furthermore, the effect on pathogenicity towards tomato was investigated. Tomato 448 infections were carried out as described above. All tested deletion strains were 449 compromised in virulence compared to WT, but the plants nevertheless developed 450 disease symptoms (Fig 7B). All infected plants exhibited stem discolorations and 451 fungal outgrowth was detected from surface sterilized stems (Fig 7C, bottom row). In this study, we identified two NLPs, Nlp2 and Nlp3, as effectors specifically secreted 554 in xylem sap. Single and double deletions of the corresponding genes resulted in 555 compromised pathogenicity on tomato. The NLP2 single deletion strain was included 556 as control as it was shown previously to contribute to V. dahliae virulence [26]. The 557 NLP3 deletion strain was additionally tested for A. thaliana root colonization revealing 558 no differences to WT (Fig 7A). This indicates that Nlp3 is dispensable for root adhesion 559 or colonization but is required during later steps of the infection.  Table). Overall, the V. dahliae genome exhibits a strikingly high

Fungal strains and growth conditions 601
Verticillium strains (S5 Table)  To identify matches of the detected peptides an in-house genome-wide protein 693 sequence database of Verticillium longisporum was used of which the protein 694 sequences are given in S1 Table. Analysis was performed by a Thermo Proteome 695

Discoverer (version 1.3) workflow that integrates Sequest and Mascot search engines. 696
For the search an initial precursor mass tolerance of 10 ppm and fragment mass 697 tolerance of 0.8 Da Carbamidomethylcysteine was used as fixed modification. 698 Oxidized methionine was included as variable modification and two miscleavages were 699 allowed for each peptide. For peptide and protein validation, a 0.5% false discovery 700 rate was set and determined by using peptide validator with a reverse decoy database. 701 Resulting lists of identified proteins were semi-quantitatively processed using the 702 Marvis Suite software [34]. Only proteins with a WoLF PSORT extracellular localization 703 prediction of above 12 were considered for further characterization [65]. A group of 704 445 identified proteins fulfilled the criteria of a threshold of 1 peptide and a high 705 intensity ratio of 0.83 in one condition. The protein sequences of the 445 proteins, and 706 details on their abundance as measured by identified peptides, are given in S1 Table.  707 The signal-to-level (s/l) ratio (see MarVis-Suite handbook on http://marvis.gobics.de) 708 was calculated using as signal the difference between SXM and xylem sap condition 709 averages (or vice versa) and as level the corresponding maximum. Polypeptides with 710 an s/l ratio above 0.3 were considered as candidates with higher intensities in the 711 specific medium and therefore belong to the specifically enriched proteins. Xylem sap-712 specific proteins are depicted in green and SXM-specific proteins in red. Whereas an 713 s/l ratio below 0.3 was considered not to be specifically enriched and formed the shared 714 core proteome.          WT/OE-GFP ∆NLP3/OE-GFP 5 dpi 3 dpi 5 dpi 3 dpi