SummarySix transcription regulatory genes of the Verticillium plant pathogen, which reprogrammed nonadherent budding yeasts for adhesion, were isolated by a genetic screen to identify control elements for early plant infection.Verticillium transcription activator of adhesion Vta2 is highly conserved in filamentous fungi but not present in yeasts. The Magnaporthe grisea ortholog conidiation regulator Con7 controls the formation of appressoria which are absent in Verticillium species. Vta2 was analyzed by using genetics, cell biology, transcriptomics, secretome proteomics and plant pathogenicity assays.Nuclear Vta2 activates the expression of the adhesin-encoding yeast flocculin genes FLO1 and FLO11. Vta2 is required for fungal growth of Verticillium where it is a positive regulator of conidiation. Vta2 is mandatory for accurate timing and suppression of microsclerotia as resting structures. Vta2 controls expression of 270 transcripts, including 10 putative genes for adhesins and 57 for secreted proteins. Vta2 controls the level of 125 secreted proteins, including putative adhesins or effector molecules and a secreted catalase-peroxidase. Vta2 is a major regulator of fungal pathogenesis, and controls host-plant root infection and H 2 O 2 detoxification.Verticillium impaired in Vta2 is unable to colonize plants and induce disease symptoms. Vta2 represents an interesting target for controlling the growth and development of these vascular pathogens.

Staphylococcus aureus is a major cause of a variety of both local and systemic infections. It can invade human host cells, a process that may account for disseminated and recurrent infections. S. aureus postinvasion events in nonprofessional phagocytes are only partially understood. While morphological data suggest a phagosomal escape, there is a lack of corroborating functional data. Using a combination of pH determination and morphological techniques, we have tested the integrity of Staphylococcus-containing phagosomes in 293 (HEK-293), HeLa, and EA.hy926 cells over time. Rapid acidification of S. aureus-containing phagosomes occurred and was sustained for up to 24 h. All S. aureus strains tested displayed equally sustained intraphagosomal pH levels without exhibiting any correlation with pH level and hemolytic activity. The membrane morphology of the phagosomal compartment was heterogeneous, even under conditions where acidic pH was fully maintained, an observation incompatible with phagolysosomal membrane destruction. As an exception, S. aureus strain 6850 showed a reduced phagosomal acidification signal 6 h after invasion. Additionally, only strain 6850 failed to localize to LAMP-1-positive vesicles in HeLa cells, although this was observed only rarely. Several other strongly beta-hemolytic strains did not modulate phagolysosomal pH, suggesting that S. aureus ␣-toxin and ␤-toxin are not sufficient for this process. Taken together, our data suggest that S. aureus-containing phagolysosomes generally remain functionally intact in nonprofessional phagocytes, thereby contrasting with transmission electron micrographic results.

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 exoproteomes of the allodiploid Verticillium longisporum upon cultivation in different media or xylem sap extracted from its host plant Brassica napus were compared. Secreted fungal proteins were identified by label free liquid chromatography-tandem mass spectrometry 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, pectinrich simulated xylem medium and pure xylem sap, which exhibited similar but additional distinct features. These exoproteomes include a shared core set of 221 secreted and similarly enriched fungal proteins. The pectin-rich medium significantly induced the secretion of 143 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 pathogenicity factors, metallopeptidases and carbohydrate-active enzymes. The most abundant 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 carbohydrate-active enzymes Gla1, Amy1 and Cbd1. Their pathogenicity contribution was analyzed in the haploid parental strain V. dahliae. Deletion of the majority of the corresponding genes caused no phenotypic changes during

23 material. Importantly, the xylem sap-specific exoproteome pinpointed Nlp2 and Nlp3 49 as single effectors required for successful V. dahliae colonization. 50 51 Author Summary 52 Verticillium spp. infect hundreds of different plants world-wide leading to enormous 53 economic losses. Verticillium wilt is a disease of the vasculature. The fungus colonizes 54 the xylem of its host plant where it exploits the vascular system to colonize the whole 55 plant. Therefore, the fungus spends part of its lifetime in this nutrient-low and 56 imbalanced environment where it is inaccessible for disease control treatments. This 57 lifestyle as well requires the fungus to react to plant defense responses by secreting 58 specific effector molecules to establish a successful infection. We addressed the 59 differences in media-dependent secretion responses of Verticillium longisporum. We 60 identified a broad response pattern induced by several media, and a similar response 61 (but with some distinct differences) for the plant-related environments: the pectin-rich 62 medium SXM and xylem sap from the host rapeseed. Importantly, we show that the 63 necrosis and ethylene inducing-like proteins Nlp2 and Nlp3 are xylem sap-specific 64 proteins that are required for full V. dahliae pathogenicity on tomato. These factors 65 play a role during the colonization phase and represent potential targets for new control 66 strategies for Verticillium wilt. 67 68 associated molecular patterns (PAMPs); examples include the fungal cell-wall polymer 95 chitin [16]. PAMP perception elicits a basal defense response which halts colonization 96 by non-adapted pathogens and results in PAMP-triggered immunity (PTI). Host-97 adapted pathogens circumvent PTI by secretion of specific effector proteins as 98 virulence factors for different phases of the infection cycle [17]. These secreted 99 effectors may act passively or actively to combat plant defense responses [18]. 100 Well known examples of fungal effectors include the Avr4 and Ecp6 effectors from the 101 leaf mold fungus Cladosporum fulvum that bind to chitin oligosaccharides via a 102 carbohydrate-binding module (CBM) or LysM domain, respectively [18-21]. Similarly, 103 a chitin scavenging function has also been assigned to Cp1 in V. dahliae strain XH-8. 104 CP1 knockout mutants were affected in cotton virulence [22]. This chitin protection 105 leads to the suppression of the PTI of the plant and shields the fungal cell wall from 106 plant chitinases that hydrolyze chitin [18-21]. Other fungal effectors such as 107 metalloproteases possess enzymatic activity and are able to truncate plant chitinases 108 that attack the fungal cell wall [23, 24]. Toxins provide another means for pathogens 109 to attack plant hosts. For example, necrosis and ethylene inducing-like proteins (NLP) 110 induce immune responses and cell death in host tissues and are conserved among 111 fungi including Verticillium spp. [25, 26]. V. dahliae isolates encode up to eight NLP 112 homologs [26, 27] whereas most other fungi gene...