Extracellular Fibrils of Pathogenic Yeast Cryptococcus gattii Are Important for Ecological Niche, Murine Virulence and Human Neutrophil Interactions

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Cryptococcus gattii, an emerging fungal pathogen of humans and animals, is found on a variety of trees in tropical and temperate regions. The ecological niche and virulence of this yeast remain poorly defined. We used Arabidopsis thaliana plants and
  Extracellular Fibrils of Pathogenic Yeast  Cryptococcus gattii   Are Important for Ecological Niche, MurineVirulence and Human Neutrophil Interactions Deborah J. Springer 1,2¤a , Ping Ren 1 , RameshRaina 3 , Yimin Dong 1 , Melissa J. Behr 1¤b ,Bruce F. McEwen 1,2 ,Samuel S. Bowser 1 , William A. Samsonoff  1 , Sudha Chaturvedi 1,2 , Vishnu Chaturvedi 1,2 * 1 Wadsworth Center, New York State Department of Health, Albany, New York, United States of America,  2 Department of Biomedical Sciences, School of Public Health,University at Albany, Albany, New York, United States of America,  3 Biology Department, Syracuse University, Syracuse, New York, United States of America Abstract Cryptococcus gattii  , an emerging fungal pathogen of humans and animals, is found on a variety of trees in tropical andtemperate regions. The ecological niche and virulence of this yeast remain poorly defined. We used  Arabidopsis thaliana plants and plant-derived substrates to model  C. gattii   in its natural habitat. Yeast cells readily colonized scratch-woundedplant leaves and formed distinctive extracellular fibrils (40–100 nm diameter 6 500–3000 nm length). Extracellular fibrilswere observed on live plants and plant-derived substrates by scanning electron microscopy (SEM) and by high voltage- EM(HVEM). Only encapsulated yeast cells formed extracellular fibrils as a capsule-deficient  C. gattii   mutant completely lackedfibrils. Cells deficient in environmental sensing only formed disorganized extracellular fibrils as apparent from experimentswith a  C. gattii STE12 a  mutant.  C. gattii   cells with extracellular fibrils were more virulent in murine model of pulmonary andsystemic cryptococcosis than cells lacking fibrils.  C. gattii   cells with extracellular fibrils were also significantly more resistantto killing by human polymorphonuclear neutrophils (PMN)  in vitro  even though these PMN produced elaborate neutrophilextracellular traps (NETs). These observations suggest that extracellular fibril formation could be a structural adaptation of C. gattii   for cell-to-cell, cell-to-substrate and/or cell-to- phagocyte communications. Such ecological adaptation of   C. gattii  could play roles in enhanced virulence in mammalian hosts at least initially via inhibition of host PMN– mediated killing. Citation:  Springer DJ, Ren P, Raina R, Dong Y, Behr MJ, et al. (2010) Extracellular Fibrils of Pathogenic Yeast  Cryptococcus gattii   Are Important for Ecological Niche,Murine Virulence and Human Neutrophil Interactions. PLoS ONE 5(6): e10978. doi:10.1371/journal.pone.0010978 Editor:  Deb Fox, The Research Institute for Children at Children’s Hospital New Orleans, United States of America Received  December 23, 2009;  Accepted  May 6, 2010;  Published  June 7, 2010 Copyright:    2010 Springer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the srcinal author and source are credited. Funding:  Partial financial support came from the National Institutes of Health (NIH) (AI-53732, AI-48462) and NIH biotechnology grant (P41-RR01219) and ClinicalLaboratory Reference System funds from the Wadsworth Center. The funders had no role in study design, data collection and analysis, decision to publish, orpreparation of the manuscript. Competing Interests:  The authors have declared that no competing interests exist.* E-mail: vishnu@wadsworth.org¤a Current address: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America¤b Current address: Wisconsin Veterinary Diagnostic Laboratory, Madison, Wisconsin, United States of America Introduction ‘Primary’ pathogenic fungi that cause serious diseases in healthyhumans occur in relatively specialized niches in nature. This traitdifferentiates them from ‘opportunistic’ fungal pathogens that afflictimmunodeficientindividuals,since ‘opportunistic’pathogenicfungiaremore widely distributed in soil and plant vegetable matter. During thelast decade, more than 400 species of ‘opportunistic’ fungi have beenrecognized as pathogens for humans and animals [1]. The broadframeworkforunderstandingrelationshipsbetweennaturaloccurrenceof the fungal pathogens and their human infectivity currently rests onthe idea of ‘dual use’ factors. It suggests that human pathogenic fungihave attributes (‘virulence factors’) that allow them to survive in soil incompetition with other life forms, to cycle through invertebrate hostssuch as amoebae and to renew their ability to infect human cells [2,3].Not specifically addressed by this idea is whether growth on plants and vegetable matter plays a role in the virulence of pathogenic fungi.Sucha scenario needs systematic investigation considering that manybacteria, especially enteric pathogens, acquire ecological fitnessthrough growth on plants [4]. Indeed, the model plant  Arabidopsis thaliana   has proven to be a tractable host for the study of bacterialpathogenesis, especially in efforts to unravel virulence factors that areshared between plant pathogens and animal pathogens [5,6]. C. gattii   is an encapsulated yeast that causes pulmonary andcerebromeningeal cryptococcosis.  C. gattii   is an emerging pathogenthat has triggered serious public health concerns due to (i) itsappearance in previously unknown geographic areas, (ii) itsoutbreaks among healthy humans, pets, and wildlife, (iii) theintractable nature of cryptococcal disease, and (iv) the difficulty of diagnosis in clinical laboratories. Reliable estimates of cryptococ-cosis due to  C. gattii   are currently lacking, but one estimate suggeststhat one-third to one-tenth of cryptococcosis cases worldwide arecaused by  C. gattii   [7]. Diagnostic laboratories routinely do notdistinguish  C. gattii   from the closely related pathogen  C. neoformans  ,since the reagents required are not readily available. Currentinformation on  C. gattii   comes either from environmental surveys,which are patchy and far between or from retrospective evaluationof   C. neoformans  / C. gattii   clinical isolates. C. gattii   is readily distinguished from closely related pathogen  C.neoformans   by occurrence on trees, rather than the pigeon PLoS ONE | www.plosone.org 1 June 2010 | Volume 5 | Issue 6 | e10978  droppings colonized by the latter. Other diagnostic characteristicsof   C. gattii   include the presence of distinctive cigar-shaped yeastmorphology in the host cerebrospinal fluid, agglutinating sero-types, creatinine assimilation and smooth, elongate, rod shapedbasidiospores of the teleomorphic form  Filobasidiella bacillispora   [8]. Any of these characteristics could serve as the starting point forsystematic studies into virulence of   C. gattii  , but other questionshave arisen. Why do rural to semi-urban forested areas posehigher risks for  C. gattii   infection? How do trees figure in theinfectious cycle? What is the nature of the infectious propagules,and how are they spread in the environment? Why are apparentlyhealthy humans and animals so prone to infection by thispathogen?To date,  C. gattii   has been associated with decayed hollows, inthe trunks and/or branches of over 50 species of angiosperms andgymnosperms. The growing body of sampling data supports theidea that  C. gattii   is long established among the fungal flora of native trees in many parts of the world [7]. Some experimentalstudies also provide evidence for  C. gattii   –plant associations.Huerfano et al. [9] demonstrated that  C. gattii   can survive onand be recovered from experimentally infected stems of almondseedlings 100 days post-inoculation, demonstrative of thispathogen’s ability to colonize and thrive on live plants. Previously,we reported that  C. gattii   will grow profusely on wood and woodextracts from a variety of trees [10]. This fungal growth washeavily melanized, which was significant as melanin is a known virulence factor. A mutation in transcription factor, Ste12 a ,caused impaired growth and loss of pigmentation on plant-basedmedia, and a concomitant loss of virulence in a murine model of cryptococcosis. These experiments suggested a link between  C. gattii   ecological fitness and virulence. Xue et al. [11] reported that C. neoformans   and  C. gattii   can colonize, cause leaf chlorosis, andproduce basidiospores on young seedlings of   A. thaliana   and Eucalyptus camaldulensis   in the laboratory; by extensions, fungalinfectious propagules are to be produced on plants in nature [11].Overall, preliminary work suggests that study of   C. gattii   –plantinteractions could elucidate unknown facets of   C. gattii   biology and virulence.We set out to develop an experimental system that capturesnative habitat and growth of wild-type  C. gattii  ; use of enrichedculture media to grow laboratory strains could mask theappearance of certain attributes, especially those that appear innature early in the establishment of infection in the humans andanimals. Further experiments focused on interactions betweenfungal cells and human phagocytes (PMN), to elucidate the factorsthat determine if the pathogen persists or is eliminated from thehost. Materials and Methods Ethics Statement Blood collection procedures from human volunteers compliedwith the New York State Department of Health InstitutionalReview Board (IRB) guidelines. Informed consent was obtainedfrom the volunteers prior to the collection of specimens. All animalstudies were conducted under full compliance with the guidelinesof the Wadsworth Center’s Institutional Animal Care and UseCommittee (IACUC) in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care(AAALAC). The protocol approval numbers were 06-333and 09-333. A daily animal welfare chart was maintained tomonitor any overt signs of pain and illness and all animals werepromptly euthanized according to recommended institutionprocedures. Fungal Strains C. gattii   NIH444 (ATCC32609; serotype B,  MAT  a  ), the wild-type strain isolated from cerebrospinal fluid (CSF), was a gift fromDr. K.J. Kwon-Chung (National Institutes of Health, Bethesda,MD). We have previously described this strain as optimum for  C. gattii   molecular pathogenesis studies [12].  C. gattii ste12 aD , amutant strain with targeted knockout of the transcription factorSTE12 a , was included as it exhibits reduced colonization of woody substrates, and reduced pathogenicity in mice as a result of defective environmental sensing [10]. We also included a capsularmutant of   C. gattii   (  cap59 D ;), because the capsule of   C. neoformans  has critical roles in fungal biology and virulence [3,13]. The  C. gattii cap59 D  mutant was constructed by homologous recombina-tion of the  cap59::NAT   disruption cassette based on sequences from C. neoformans CAP59  [14]. The PCR amplifications, biolistictransformation and confirmation of acapsular morphology wasaccording to standard methods [10,15,16]. Growth Media C. gattii   cells were cultured in YPD broth at 30 u C with shaking (180 rpm), and were maintained in YPD agar and preserved at 2 70 u C in 15% sterile glycerol or in liquid N 2 . Un- treated black cherry (  Prunus serotina   ) wood chips were obtained from RogerDziengeleski, Finch, Pruyn & Co., Inc. (Glens Falls, NY). Leaf agar was prepared by mincing of 10 g of   A. thaliana   leaves intosmall pieces with scissors, and addition of 2% agar with 0.1%glucose followed by autoclaving for 15 min at 121 u C [10].  Arabidopsis thaliana  A. thaliana   ecotype Columbia (Col-0) and Landsberg erecta (Ler-0), and various mutant lines  eds1  (enhanced disease susceptibility 1;lipase/signal transducer/triacylglycerol lipase),  nahG   (transgenicline degrading salicylic acid; SA),  npr1  (nonexpressor of PR genes1; pathogenesis-related 1),  sid1  (SA-induction deficient),  rpm1 (resistance to  Pseudomonas syringae   pv maculicola 1), were grown in agreenhouse at the Biology Department, Syracuse University,Syracuse, NY [17,18,19,20]. Four- to six-week old plants weretransferred to the Mycology Laboratory of the Wadsworth Centerwhere they were maintained at 20–23 u C with a 12 hr light/dark cycle under 50–70% humidity, in a modified incubator withHEPA filtration. Plant inoculation, harvest and light microscopy C. gattii   cells were subcultured twice in YPD broth at 30 u C with180 rpm shaking and were then collected by centrifugation (3 minat 3,800 rpm), washed twice in deionized sterile water (DSW) andre-suspended to a final concentration of 1.0 6 10 6 cells/ml, bycounting in a hemacytometer.  A. thaliana   plants from the growthchamber were placed in a sealed carrier case and moved to a BSL-2 safety cabinet. Four to six leaves on each  A. thaliana   plant weremarked with a glass marker. Leaves were lightly wounded on theadaxial leaf surface with a 2–3 mm shallow scratch (withoutcomplete puncture) on either side of the mid-vein with a 27-gaugesyringe needle [21,22,23]. Two 5- m l drops of 10 6 C. gattii   cells/mlwere placed at the wound site, and allowed to air dry (5–10 min).Plants were replaced in modified growth incubator maintained at20–23 u C, 12 hr light/dark cycle, and 50–70% humidity. Macro-photographic images of   A. thaliana   plants and leaves were obtainedwith a consumer digital camera.Infected  A. thaliana   leaves were chosen at random and cut fromthe plant 7 days post-inoculation and homogenized with a tissuegrinder in 1 ml of DSW, and the homogenate was serially diluted.100- m L of each dilution was plated onto YPD agar and incubated C. gattii   FibrilsPLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e10978  for 3–5 days at 25 u C, and colonies were counted for determinationof fungal viability.For light microscopy, leaves were cut from plants 7 or 14 dayspost-inoculation, fixed in 2% glutaraldehyde (EM grade) inphosphate-buffered saline (PBS) or in 0.2 M sodium cacodylatebuffer, pH 7.4, alcohol dehydrated by graded series (25%, 50%,75%, 95% 100%), and stored for further processing in 100%ethanol at 4 u C [24]. Staining of whole leaves was accomplished bysoaking of leaves in an aqueous solution of equal parts of Trypanblue (1 mg/ml), lactic acid, deionized water, and glycerol for 4-min [25]. Leaves were destained in chloral hydrate (2.5 g/ml),slide-mounted, and viewed and imaged with a table top scannerand light microscope. Growth on plant-based and artificial substrates  A number of natural and artificial substrates were used to studygrowth characteristics of   C. gattii  . The natural substrates wereintended to recapitulate growth of   C. gattii   on plants in nature, andfor comparisons to growth on organic or synthetic substrates.Wild-type and  C. gattii cap59 D  mutant strains were grown to mid-logarithmic phase in YPD broth. Cells were collected bycentrifugation, washed two times in minimal asparagine medium,with 1% glucose, and re-suspended in the same medium to a finalconcentration of 1 6 10 6 cells/ml. Wild-type  C. gattii cap59 D mutant cells were treated with sodium azide (2 mM), followedby incubation at 65 u C for 30 minutes to render them non-viable;these cells were used in experiments that compared effects of  viable and non-viable cells. The plant-based and artificialsubstrates used were: autoclaved black cherry wood shavings(Finch, Pruyn & Co., Inc., Glens Falls, NY), consumer brownpaper bag, Whatman  # 1 qualitative filter paper with 98%cellulose (Whatman Limited, Kent, England), Thermanox H  plasticcoverslips surface treated for optimal cell adhesion (Nalge NuncInternational, Rochester, NY), and glass coverslip (Ted Pella, Inc.,Redding, CA) and dialysis membrane. The substrate was placed atthe bottom of 18-mm round wells of a 24-well flat-bottompolystyrene cell culture plate. One milliliter of 1 6 10 6 cells inminimal asparagine medium, was added to each well in one row of the 24 wells in duplicate. The culture plates were covered andincubated for 24- or 48-hr at 25 u C. The cell suspension wasremoved, and wells were washed 3 times with 0.05% Tween-80 inTris-buffered saline (TBS), to remove non-adherent cells. Sub-strates were fixed with 2% glutaraldehyde in 0.2 M sodiumcacodylate buffer, pH 7.4 for 24 hr at 4 u C, and dehydrated byexposure to a graded ethanol-water series, and stored in 100%ethanol in plates. Average numbers of attached cells weredetermined by counting cells in four SEM fields per sample. Scanning electron microscopy (SEM)  A. thaliana   leaves inoculated with  C. gattii   were harvested atindicated intervals, fixed in 2% glutaraldehyde (EM grade) inphosphate-buffered saline (PBS) or 0.2 M sodium cacodylatebuffer, pH 7.4, alcohol-dehydrated by graded series (25%, 50%,75%, 95%, 100%), and stored in 100% ethanol. The dehydratedspecimens were critical point dried with liquid CO 2  in a tousimisSamdri 795 drier (Rockville, MD, USA), gold-sputter-coated,observed and imaged with a LEO 1550 Variable Pressure fieldemission gun SEM (Carl Zeiss SMT, Peabody, MA), according toprocedures followed in our laboratory [24]. A semi-quantitativeestimate of   C. gattii   attachment to the natural or artificial substrateswas obtained similarly to our quantification of mating in SEMimages described previously [10]. Briefly, cells were counted bothindividually and in clusters. A cell cluster was defined as anaggregation of four or more  C. gattii   cells. Mean average countswere obtained from determining the number of   C. gattii   cells in atleast four or more SEM images. High voltage electron microscopy (HVEM)  A. thaliana   leaves that have been scratch-wounded andinoculated with  C. gattii   cells were harvested and fixed in 2%glutaraldehyde (EM grade) in 0.2 M sodium cacodylate buffer,pH 7.4 overnight. Leaves were washed three times with 0.2 Msodium cacodylate buffer and stained with 1% osmium tetroxide(OsO 4  ) in 0.1 M sodium cacodylate for 1-hr at 4 u C, rinsed twotimes with DSW, and dehydrated through a graded ethanol series[26]. Samples were then embedded in Spurr low-viscosityembedding medium (Polysciences, Inc.; Warrington, PA, USA),which was allowed to polymerize at 70 u C for 48-hr. Blocks withembedded  A. thaliana   leaves were cut and trimmed with a hot razorblade, and mounted on pre-formed TEM pegs. Sections were cutapproximately 1  m m thick, on a microtome and were mounted onformvar-coated grids. Grid-mounted sections were then stainedwith 2% uranyl acetate for 60 min, followed by Reynold’s leadstain for 20 min; they were then allowed to air dry, and werestored in an EM grid box. Sections were observed and imagedwith the 1.2 MeV AEI EM7 MK II high voltage electronmicroscope (HVEM) at the Wadsworth Center. Effects of inhibitors of cytoskeletal proteins We wanted to examine the roles of actin, tubulins and othercytoskeletal proteins on  C. gattii   microtubes, since these proteinsare important determinats of cell shape [27,28].  C. gattii   wild-typeand  cap59 D  mutant strain were grown in YPD broth, 30 u C,180 rpm for 12–16 hr and treated with thiabendazole, cytocha-lasin B, mebendazol, or latrunculin B (details in Suppl.information). Murine model of cryptococcosis Virulence of   C. gattii   was tested in a murine model of cryptococcosis that mimics either progressive pulmonary crypto-coccosis (intranasal inoculation, IN) or rapid onset of cerebrome-ningeal cryptococcosis (intravenous inoculation, IV) as describedpreviously [10,29,30].  C. gattii   cells were grown with two successivepassages on YPD agar and  A. thaliana   leaf-agar at 25 u C for 4 days.Four- to six-week old male BALB/c mice were obtained fromCharles River Laboratories, Inc. Groups of 8 BALB/c mice wereinoculated either via IN or IV route with 30  m l or 100  m l DSWcontaining 10 5 cells or 10 4 cells, respectively. Mice were given foodand water  ad libitum , and were observed daily according to awelfare score sheet that addresses appearance of diseasesymptoms, general malaise, and any apparent pain and discomfortin the infected animals. After signs of progressive poor health ordiscomfort were noted, the infected animals were euthanized byCO 2  inhalation followed by cervical dislocation. Data from fiveinfected mice (IN or IV) were used to determine Kaplan-Meyersurvival curve using SAS software (SAS Institute, Inc.; Cary, N.C). An assessment of colonization and multiplication within lungs andbrain of infected mice was obtained by using three mice from eachgroup, which were euthanized at day 7 (IV) or day 14 (IN) postinfection. None of the animals displayed any signs of overt illnessat these two time periods. One-half of lung or brain harvestedfrom infected animals was preserved for histopathological analyses,in Bouin’s fixative (brain section) or buffered formalin (lungs). Theother halves of target organs were used to calculate relative fungalloads. Tissues were weighed and homogenized in 1 ml DSW,serial dilutions were plated on YPD agar, and incubated at 30 u Cfor determination of CFU. Histopathological examinations weredone as described previously [10]. Briefly, after 24 hr of fixation, C. gattii   FibrilsPLoS ONE | www.plosone.org 3 June 2010 | Volume 5 | Issue 6 | e10978  tissues were sectioned, and brain sections were washed in distilledwater for several hours. Processing was done in a vacuuminfiltration processor, the Tissue-Tek VIP 5 (Sakura Finetech),starting with 70% ethanol and proceeding through a series of dehydrating alcohols and xylenes into paraffin, for 15 min perstation. Tissues were then embedded in paraffin blocks andsectioned at a 7- m m (brain tissues) and 3–4  m m (lung tissues)thickness. Sections were stained with hematoxylin & eosin andmucicarmine (Richard Allen Scientific) and examined by lightmicroscopy. C. gattii  –PMN interactions We have previously shown that  in vitro  interactions betweenhuman PMN and  C. gattii   can be used for a complimentaryevaluation of virulence outcome in mouse models, and to assessthe likely outcome of initial interactions between  C. gattii   andphagocyte cells [10,29,31]. We compared  C. gattii   cells grown onleaf agar to  C. gattii   cells grown on YPD agar; a clinical isolate of  Candida glabrata   was used as control as was  C. gattii cap59 D  mutant.Blood was obtained from human volunteers per protocol approvedby the New York State Department of Health Institutional ReviewBoard. PMN were isolated by Ficol-Paque (Pharmacia LKBBiotechnology) centrifugation [10,29,30]. PMN were washed twiceand resuspended in RPMI-1640. PMN and yeast cells wereincubated at a 10:1 ratio for 4 hr at 37 u C under 5% CO 2 , followedby plating on YPD agar for CFU determination [31]. Percentfungicidal activity was calculated as: 1-(CFU experiment  4  CFUcontrols) 6 100. Results were expressed as means 6 standard error(SE) from the values from at least four individual blood donors[10,29]. SEM analysis of   C. gattii  -PMN interactions were carriedusing Thermanox H  plastic cover slips in 24-well flat-bottom tissueculture plates. PMNs and yeast cells were mixed at a 1:10 ratio,added to each well, and incubated for 4 hr at 37 u C under 5%CO 2 . Following incubation, cells were fixed with 2% glutaralde-hyde and processed as described earlier. Results C. gattii   proliferates on  A. thaliana  leaves Gross examination of   A. thaliana   ecotype Col-0 leaves inoculatedwith  C. gattii   wild-type strain showed noticeable chlorotic lesionsaround the initial wound site. Leaves inoculated with  C. gattii cap59 D  mutant strain had smaller chlorotic lesions (Figs. 1D, 1G).However, neither of two  C. gattii   strains caused systemic plantdisease in contrast to what has been reported for a number of plantpathogenic fungi grown on  A. thaliana   plants [32,33]. Microscopicexaminations of trypan blue-stained leaves for the presence of  yeast cells showed  C. gattii   colonization along wound sites, as wellas punctuated colonization across the leaf, even distant from theinitial inoculum site and wound (Fig. 1E,1F). In contrast,  A. thaliana  leaves inoculated with  C. gattii cap59 D  mutant had much lowernumber of yeast cells that were confined to the initial inoculation Figure 1.  C. gattii   colonized experimentally inoculated  A. thaliana   leaves.  A. thaliana  leaves on live plants were inoculated with steriledeionized water control (A),  C. gattii   wild type cells (D), or  C. gattii cap59 D  mutant (G). Gross examination of the control leaves with mock inoculationsshowed no scars while large chlorotic lesions were seen in leaves inoculated with  C. gattii   wild type cells; much smaller lesions were visible in leavesinoculated with  C. gattii   mutant cells. Further examination of inoculated leaves after fixation and staining showed no significant fungal colonizationaround inoculation sites in the mock- inoculated and  cap59 D  mutant-inoculated leaves (B, H), but leaves inoculated with  C. gattii   wild-type cellsshowed fungal cells around inoculation sites (E). Microscopic examinations of leaves revealed numerous  C. gattii   wild-type cells at the inoculation siteand also cells that had dispersed away from the srcinal inoculation (F); in contrast, cap59 D  mutant cells were localized at few spots around theinoculation site (I) (100 6 magnification). Panels A, B and C are from different leaves to illustrate salient features of these observations.doi:10.1371/journal.pone.0010978.g001 C. gattii   FibrilsPLoS ONE | www.plosone.org 4 June 2010 | Volume 5 | Issue 6 | e10978  sites (Figs. 1H and 1I). Control leaves with mock-inoculation didnot display any specific staining (Fig. 1B–1C). Yeast cells werestained most heavily at the wound edges in the leaves consistentwith observed chlorotic regions (Fig. 1E). Overall, there was ampleevidence for  C. gattii   wild-type strain colonization and dispersalfrom the wound sites on  A. thaliana   Col-0 ecotype leaves.Preliminary examination of another ecotype  A. thaliana   Ler-0indicated almost two-times more colonization visually as seen onCol-0 ecotype (Fig. S1). Additionally, a number of mutant plantlines derived from  A. thaliana   Col-0 ecotype and deficient in plantdefense against fungal pathogens, showed higher colonization of inoculated leaves by  C. gattii   wild-type strain (  eds1 . nahG  . sid2 . npr1 . rpm1 ; Fig. S2). C. gattii   extracellular fibrils on plant leaves Further examinations by SEM revealed that  C. gattii   profuselycolonized wounded  A. thaliana   leaves, with the highest numbers of  yeast cells present at the wound sites, but significant numbers of cellsseen away from the wounds, a pattern consistent with the lightmicroscopic observations (Fig. 2A, B). Although  C. gattii   cells onleaves were intact, and showed buds of various sizes, we could nottell whether colonization away from wound sites resulted by active Figure 2.  C. gattii   formed extracellular fibrils on  A. thaliana   leaves.  SEM images of infected leaves showed that  C. gattii   colonized wound siteand crevices along leaf surfaces (A, B), extracellular fibrils were visible projecting from  C. gattii   cells, connecting yeast cells to each other and to theleaf surface (C, D). Note a yeast cell at higher magnification with prominent extracellular fibrils extending to other yeast cells and to the leaf tissue (D).Higher magnification of   C. gattii  - inoculated leaf surface revealed formation of ‘leaf halo; and ‘pocket’ in  A. thaliana  in some instances (E, F). Scale barequals 100  m m (A), 5.0  m m (B, C), and 1.0  m m (D, E, F).doi:10.1371/journal.pone.0010978.g002 C. gattii   FibrilsPLoS ONE | www.plosone.org 5 June 2010 | Volume 5 | Issue 6 | e10978
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