A receptor pair with an integrated decoy converts pathogen disabling of transcription factors to immunity.

Microbial pathogens infect host cells by delivering virulence factors (effectors) that interfere with defenses. In plants, intracellular nucleotide-binding/leucine-rich repeat receptors (NLRs) detect specific effector interference and trigger immunity by an unknown mechanism. The Arabidopsis-interacting NLR pair, RRS1-R with RPS4, confers resistance to different pathogens, including Ralstonia solanacearum bacteria expressing the acetyltransferase effector PopP2. We show that PopP2 directly acetylates a key lysine within an additional C-terminal WRKY transcription factor domain of RRS1-R that binds DNA. This disrupts RRS1-R DNA association and activates RPS4-dependent immunity. PopP2 uses the same lysine acetylation strategy to target multiple defense-promoting WRKY transcription factors, causing loss of WRKY-DNA binding and transactivating functions needed for defense gene expression and disease resistance. Thus, RRS1-R integrates an effector target with an NLR complex at the DNA to switch a potent bacterial virulence activity into defense gene activation.

Correction: Autoacetylation of the Ralstonia solanacearum Effector PopP2 Targets a Lysine Residue Essential for RRS1-R-Mediated Immunity in Arabidopsis.

[This corrects the article DOI: 10.1371/journal.ppat.1001202.].

An oomycete effector targets a plant RNA helicase involved in root development and defense.

Oomycete plant pathogens secrete effector proteins to promote disease. The damaging soilborne legume pathogen Aphanomyces euteiches harbors a specific repertoire of Small Secreted Protein effectors (AeSSPs), but their biological functions remain unknown. Here we characterize AeSSP1256. The function of AeSSP1256 is investigated by physiological and molecular characterization of Medicago truncatula roots expressing the effector. A potential protein target of AeSSP1256 is identified by yeast-two hybrid, co-immunoprecipitation, and fluorescent resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) assays, as well as promoter studies and mutant characterization. AeSSP1256 impairs M. truncatula root development and promotes pathogen infection. The effector is localized to the nucleoli rim, triggers nucleoli enlargement and downregulates expression of M. truncatula ribosome-related genes. AeSSP1256 interacts with a functional nucleocytoplasmic plant RNA helicase (MtRH10). AeSSP1256 relocates MtRH10 to the perinucleolar space and hinders its binding to plant RNA. MtRH10 is associated with ribosome-related genes, root development and defense. This work reveals that an oomycete effector targets a plant RNA helicase, possibly to trigger nucleolar stress and thereby promote pathogen infection.

The rhizobial type III effector ErnA confers the ability to form nodules in legumes.

Several Bradyrhizobium species nodulate the leguminous plant Aeschynomene indica in a type III secretion system-dependent manner, independently of Nod factors. To date, the underlying molecular determinants involved in this symbiotic process remain unknown. To identify the rhizobial effectors involved in nodulation, we mutated 23 out of the 27 effector genes predicted in Bradyrhizobium strain ORS3257. The mutation of nopAO increased nodulation and nitrogenase activity, whereas mutation of 5 other effector genes led to various symbiotic defects. The nopM1 and nopP1 mutants induced a reduced number of nodules, some of which displayed large necrotic zones. The nopT and nopAB mutants induced uninfected nodules, and a mutant in a yet-undescribed effector gene lost the capacity for nodule formation. This effector gene, widely conserved among bradyrhizobia, was named ernA for "effector required for nodulation-A." Remarkably, expressing ernA in a strain unable to nodulate A. indica conferred nodulation ability. Upon its delivery by Pseudomonas fluorescens into plant cells, ErnA was specifically targeted to the nucleus, and a fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy approach supports the possibility that ErnA binds nucleic acids in the plant nuclei. Ectopic expression of ernA in A. indica roots activated organogenesis of root- and nodule-like structures. Collectively, this study unravels the symbiotic functions of rhizobial type III effectors playing distinct and complementary roles in suppression of host immune functions, infection, and nodule organogenesis, and suggests that ErnA triggers organ development in plants by a mechanism that remains to be elucidated.

Direct and Indirect Visualization of Bacterial Effector Delivery into Diverse Plant Cell Types during Infection.

To cause disease, diverse pathogens deliver effector proteins into host cells. Pathogen effectors can inhibit defense responses, alter host physiology, and represent important cellular probes to investigate plant biology. However, effector function and localization have primarily been investigated after overexpression in planta. Visualizing effector delivery during infection is challenging due to the plant cell wall, autofluorescence, and low effector abundance. Here, we used a GFP strand system to directly visualize bacterial effectors delivered into plant cells through the type III secretion system. GFP is a beta barrel that can be divided into 11 strands. We generated transgenic Arabidopsis thaliana plants expressing GFP1-10 (strands 1 to 10). Multiple bacterial effectors tagged with the complementary strand 11 epitope retained their biological function in Arabidopsis and tomato (Solanum lycopersicum). Infection of plants expressing GFP1-10 with bacteria delivering GFP11-tagged effectors enabled direct effector detection in planta. We investigated the temporal and spatial delivery of GFP11-tagged effectors during infection with the foliar pathogen Pseudomonas syringae and the vascular pathogen Ralstonia solanacearum Thus, the GFP strand system can be broadly used to investigate effector biology in planta.

A two genes - for - one gene interaction between Leptosphaeria maculans and Brassica napus.

Interactions between Leptosphaeria maculans, causal agent of stem canker of oilseed rape, and its Brassica hosts are models of choice to explore the multiplicity of 'gene-for-gene' complementarities and how they diversified to increased complexity in the course of plant-pathogen co-evolution. Here, we support this postulate by investigating the AvrLm10 avirulence that induces a resistance response when recognized by the Brassica nigra resistance gene Rlm10. Using genome-assisted map-based cloning, we identified and cloned two AvrLm10 candidates as two genes in opposite transcriptional orientation located in a subtelomeric repeat-rich region of the genome. The AvrLm10 genes encode small secreted proteins and show expression profiles in planta similar to those of all L. maculans avirulence genes identified so far. Complementation and silencing assays indicated that both genes are necessary to trigger Rlm10 resistance. Three assays for protein-protein interactions showed that the two AvrLm10 proteins interact physically in vitro and in planta. Some avirulence genes are recognized by two distinct resistance genes and some avirulence genes hide the recognition specificities of another. Our L. maculans model illustrates an additional case where two genes located in opposite transcriptional orientation are necessary to induce resistance. Interestingly, orthologues exist for both L. maculans genes in other phytopathogenic species, with a similar genome organization, which may point to an important conserved effector function linked to heterodimerization of the two proteins.

Recruitment of a Lineage-Specific Virulence Regulatory Pathway Promotes Intracellular Infection by a Plant Pathogen Experimentally Evolved into a Legume Symbiont.

Ecological transitions between different lifestyles, such as pathogenicity, mutualism and saprophytism, have been very frequent in the course of microbial evolution, and often driven by horizontal gene transfer. Yet, how genomes achieve the ecological transition initiated by the transfer of complex biological traits remains poorly known. Here, we used experimental evolution, genomics, transcriptomics and high-resolution phenotyping to analyze the evolution of the plant pathogen Ralstonia solanacearum into legume symbionts, following the transfer of a natural plasmid encoding the essential mutualistic genes. We show that a regulatory pathway of the recipient R. solanacearum genome involved in extracellular infection of natural hosts was reused to improve intracellular symbiosis with the Mimosa pudica legume. Optimization of intracellular infection capacity was gained through mutations affecting two components of a new regulatory pathway, the transcriptional regulator efpR and a region upstream from the RSc0965-0967 genes of unknown functions. Adaptive mutations caused the downregulation of efpR and the over-expression of a downstream regulatory module, the three unknown genes RSc3146-3148, two of which encoding proteins likely associated to the membrane. This over-expression led to important metabolic and transcriptomic changes and a drastic qualitative and quantitative improvement of nodule intracellular infection. In addition, these adaptive mutations decreased the virulence of the original pathogen. The complete efpR/RSc3146-3148 pathway could only be identified in the genomes of the pathogenic R. solanacearum species complex. Our findings illustrate how the rewiring of a genetic network regulating virulence allows a radically different type of symbiotic interaction and contributes to ecological transitions and trade-offs.

Immunity at Cauliflower Hydathodes Controls Systemic Infection by Xanthomonas campestris pv campestris.

Hydathodes are water pores found on leaves of a wide range of vascular plants and are the sites of guttation. We report here on the detailed anatomy of cauliflower (Brassicaoleracea) and Arabidopsis (Arabidopsis thaliana) hydathodes. Hydathode surface presents pores resembling stomata giving access to large cavities. Beneath, the epithem is composed of a lacunar and highly vascularized parenchyma offering a direct connection between leaf surface and xylem vessels. Arabidopsis hydathode pores were responsive to ABA and light similar to stomata. The flg22 flagellin peptide, a well-characterized elicitor of plant basal immunity, did not induce closure of hydathode pores in contrast to stomata. Because hydathodes are natural infection routes for several pathogens, we investigated hydathode infection by the adapted vascular phytopathogenic bacterium Xanthomonas campestris pv campestris (Xcc), the causal agent of black rot disease of Brassicaceae. Microscopic observations of hydathodes six days postinoculation indicated a digestion of the epithem cells and a high bacterial multiplication. Postinvasive immunity was shown to limit pathogen growth in the epithem and is actively suppressed by the type III secretion system and its effector proteins. Altogether, these results give a detailed anatomic description of Brassicaceae hydathodes and highlight the efficient use of this tissue as an initial niche for subsequent vascular systemic dissemination of Xcc in distant plant tissues.

Functional Analysis of γ-Tubulin Complex Proteins Indicates Specific Lateral Association via Their N-terminal Domains.

Microtubules are nucleated from multiprotein complexes containing γ-tubulin and associated γ-tubulin complex proteins (GCPs). Small complexes (γTuSCs) comprise two molecules of γ-tubulin bound to the C-terminal domains of GCP2 and GCP3. γTuSCs associate laterally into helical structures, providing a structural template for microtubule nucleation. In most eukaryotes γTuSCs associate with additional GCPs (4, 5, and 6) to form the core of the so-called γ-tubulin ring complex (γTuRC). GCPs 2-6 constitute a family of homologous proteins. Previous structural analysis and modeling of GCPs suggest that all family members can potentially integrate into the helical structure. Here we provide experimental evidence for this model. Using chimeric proteins in which the N- and C-terminal domains of different GCPs are swapped, we show that the N-terminal domains define the functional identity of GCPs, whereas the C-terminal domains are exchangeable. FLIM-FRET experiments indicate that GCP4 and GCP5 associate laterally within the complex, and their interaction is mediated by their N-terminal domains as previously shown for γTuSCs. Our results suggest that all GCPs are incorporated into the helix via lateral interactions between their N-terminal domains, whereas the C-terminal domains mediate longitudinal interactions with γ-tubulin. Moreover, we show that binding to γ-tubulin is not essential for integrating into the helical complex.

The Eucalyptus linker histone variant EgH1.3 cooperates with the transcription factor EgMYB1 to control lignin biosynthesis during wood formation.

Wood, also called secondary xylem, is a specialized vascular tissue constituted by different cell types that undergo a differentiation process involving deposition of thick, lignified secondary cell walls. The mechanisms needed to control the extent of lignin deposition depending on the cell type and the differentiation stage are far from being fully understood. We found that the Eucalyptus transcription factor EgMYB1, which is known to repress lignin biosynthesis, interacts specifically with a linker histone variant, EgH1.3. This interaction enhances the repression of EgMYB1's target genes, strongly limiting the amount of lignin deposited in xylem cell walls. The expression profiles of EgMYB1 and EgH1.3 overlap in xylem cells at early stages of their differentiation as well as in mature parenchymatous xylem cells, which have no or only thin lignified secondary cell walls. This suggests that a complex between EgMYB1 and EgH1.3 integrates developmental signals to prevent premature or inappropriate lignification of secondary cell walls, providing a mechanism to fine-tune the differentiation of xylem cells in time and space. We also demonstrate a role for a linker histone variant in the regulation of a specific developmental process through interaction with a transcription factor, illustrating that plant linker histones have other functions beyond chromatin organization.

Experimental evolution of rhizobia may lead to either extra- or intracellular symbiotic adaptation depending on the selection regime.

Experimental evolution is a powerful approach to study the process of adaptation to new environments, including the colonization of eukaryotic hosts. Facultative endosymbionts, including pathogens and mutualists, face changing and spatially structured environments during the symbiotic process, which impose diverse selection pressures. Here, we provide evidence that different selection regimes, involving different times spent in the plant environment, can result in either intra- or extracellular symbiotic adaptations. In previous work, we introduced the symbiotic plasmid of Cupriavidus taiwanensis, the rhizobial symbiont of Mimosa pudica, into the phytopathogen Ralstonia solanacearum and selected three variants able to form root nodules on M. pudica, two (CBM212 and CBM349) being able to rudimentarily infect nodule cells and the third one (CBM356) only capable of extracellular infection of nodules. Each nodulating ancestor was further challenged to evolve using serial ex planta-in planta cycles of either 21 (three short-cycle lineages) or 42 days (three long-cycle lineages). In this study, we compared the phenotype of the 18 final evolved clones. Evolution through short and long cycles resulted in similar adaptive paths on lineages deriving from the two intracellularly infectious ancestors, CBM212 and CBM349. In contrast, only short cycles allowed a stable acquisition of intracellular infection in lineages deriving from the extracellularly infecting ancestor, CBM356. Long cycles, instead, favoured improvement of extracellular infection. Our work highlights the importance of the selection regime in shaping desired traits during host-mediated selection experiments.

Exploring fungus-plant N transfer in a tripartite ant-plant-fungus mutualism.

Background and Aims: The plant Hirtella physophora, the ant Allomerus decemarticulatus and a fungus, Trimmatostroma sp., form a tripartite association. The ants manipulate both the plant trichomes and the fungus to build galleries under the stems of their host plant used to capture prey. In addition to its structural role, the fungus also improves nutrient uptake by the host plant. But it still remains unclear whether the fungus plays an indirect or a direct role in transferring nutrients to the plant. This study aimed to trace the transfer of N from the fungus to the plant's stem tissue. Methods: Optical microscopy and transmission electron microscopy (TEM) were used to investigate the presence of fungal hyphae in the stem tissues. Then, a 15N-labelling experiment was combined with a nanoscale secondary-ion mass spectrometry (NanoSIMS 50) isotopic imaging approach to trace the movement of added 15N from the fungus to plant tissues. Key Results: The TEM images clearly showed hyphae inside the stem tissue in the cellular compartment. Also, fungal hyphae were seen perforating the wall of the parenchyma cell. The 15N provisioning of the fungus in the galleries resulted in significant enrichment of the 15N signature of the plant's leaves 1 d after the 15N-labelling solution was deposited on the fungus-bearing trap. Finally, NanoSIMS imaging proved that nitrogen was transferred biotrophically from the fungus to the stem tissue. Conclusions: This study provides evidence that the fungi are connected endophytically to an ant-plant system and actively transfer nitrogen from 15N-labelling solution to the plant's stem tissues. Overall, this study underlines how complex the trophic structure of ant-plant interactions is due to the presence of the fungus and provides insight into the possibly important nutritional aspects and tradeoffs involved in myrmecophyte-ant mutualisms.

Calcium- and Nitric Oxide-Dependent Nuclear Accumulation of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Response to Long Chain Bases in Tobacco BY-2 Cells.

Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid long chain bases (LCBs) in plants, is known to induce a calcium-dependent programmed cell death (PCD) in plants. In addition, in tobacco BY-2 cells, it has been shown that DHS triggers a rapid production of H2O2 and nitric oxide (NO). Recently, in analogy to what is known in the animal field, plant cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC), a ubiquitous enzyme involved in glycolysis, has been suggested to fulfill other functions associated with its oxidative post-translational modifications such as S-nitrosylation on cysteine residues. In particular, in mammals, stress signals inducing NO production promote S-nitrosylation of GAPC and its subsequent translocation into the nucleus where the protein participates in the establishment of apoptosis. In the present study, we investigated the behavior of GAPC in tobacco BY-2 cells treated with DHS. We found that upon DHS treatment, an S-nitrosylated form of GAPC accumulated in the nucleus. This accumulation was dependent on NO production. Two genes encoding GAPCs, namely Nt(BY-2)GAPC1 and Nt(BY-2)GAPC2, were cloned. Transient overexpression of Nt(BY-2)GAPC-green fluorescent protein (GFP) chimeric constructs indicated that both proteins localized in the cytoplasm as well as in the nucleus. Mutating into serine the two cysteine residues thought to be S-nitrosylated in response to DHS did not modify the localization of the proteins, suggesting that S-nitrosylation of GAPCs was probably not necessary for their nuclear relocalization. Interestingly, using Förster resonance energy transfer experiments, we showed that Nt(BY-2)GAPCs interact with nucleic acids in the nucleus. When GAPCs were mutated on their cysteine residues, their interaction with nucleic acids was abolished, suggesting a role for GAPCs in the protection of nucleic acids against oxidative stress.

CRN13 candidate effectors from plant and animal eukaryotic pathogens are DNA-binding proteins which trigger host DNA damage response.

To successfully colonize their host, pathogens produce effectors that can interfere with host cellular processes. Here we investigated the function of CRN13 candidate effectors produced by plant pathogenic oomycetes and detected in the genome of the amphibian pathogenic chytrid fungus Batrachochytrium dendrobatidis (BdCRN13). When expressed in Nicotiana, AeCRN13, from the legume root pathogen Aphanomyces euteiches, increases the susceptibility of the leaves to the oomycete Phytophthora capsici. When transiently expressed in amphibians or plant cells, AeCRN13 and BdCRN13 localize to the cell nuclei, triggering aberrant cell development and eventually causing cell death. Using Förster resonance energy transfer experiments in plant cells, we showed that both CRN13s interact with nuclear DNA and trigger plant DNA damage response (DDR). Mutating key amino acid residues in a predicted HNH-like endonuclease motif abolished the interaction of AeCRN13 with DNA, the induction of DDR and the enhancement of Nicotiana susceptibility to P. capsici. Finally, H2AX phosphorylation, a marker of DNA damage, and enhanced expression of genes involved in the DDR were observed in A. euteiches-infected Medicago truncatula roots. These results show that CRN13 from plant and animal eukaryotic pathogens promotes host susceptibility by targeting nuclear DNA and inducing DDR.

The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding.

Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect binding and are multidomain proteins generally carrying a nucleotide binding (NB) and a leucine-rich repeat (LRR) domain. Two NB-LRR protein-coding genes from rice (Oryza sativa), RGA4 and RGA5, were found to be required for the recognition of the Magnaporthe oryzae effector AVR1-CO39. RGA4 and RGA5 also mediate recognition of the unrelated M. oryzae effector AVR-Pia, indicating that the corresponding R proteins possess dual recognition specificity. For RGA5, two alternative transcripts, RGA5-A and RGA5-B, were identified. Genetic analysis showed that only RGA5-A confers resistance, while RGA5-B is inactive. Yeast two-hybrid, coimmunoprecipitation, and fluorescence resonance energy transfer-fluorescence lifetime imaging experiments revealed direct binding of AVR-Pia and AVR1-CO39 to RGA5-A, providing evidence for the recognition of multiple Avr proteins by direct binding to a single R protein. Direct binding seems to be required for resistance as an inactive AVR-Pia allele did not bind RGA5-A. A small Avr interaction domain with homology to the Avr recognition domain in the rice R protein Pik-1 was identified in the C terminus of RGA5-A. This reveals a mode of Avr protein recognition through direct binding to a novel, non-LRR interaction domain.

Shaping bacterial symbiosis with legumes by experimental evolution.

Nitrogen-fixing symbionts of legumes have appeared after the emergence of legumes on earth, approximately 70 to 130 million years ago. Since then, symbiotic proficiency has spread to distant genera of α- and ß-proteobacteria, via horizontal transfer of essential symbiotic genes and subsequent recipient genome remodeling under plant selection pressure. To tentatively replay rhizobium evolution in laboratory conditions, we previously transferred the symbiotic plasmid of the Mimosa symbiont Cupriavidus taiwanensis in the plant pathogen Ralstonia solanacearum, and selected spontaneous nodulating variants of the chimeric Ralstonia sp. using Mimosa pudica as a trap. Here, we pursued the evolution experiment by submitting two of the rhizobial drafts to serial ex planta-in planta (M. pudica) passages that may mimic alternating of saprophytic and symbiotic lives of rhizobia. Phenotyping 16 cycle-evolved clones showed strong and parallel evolution of several symbiotic traits (i.e., nodulation competitiveness, intracellular infection, and bacteroid persistence). Simultaneously, plant defense reactions decreased within nodules, suggesting that the expression of symbiotic competence requires the capacity to limit plant immunity. Nitrogen fixation was not acquired in the frame of this evolutionarily short experiment, likely due to the still poor persistence of final clones within nodules compared with the reference rhizobium C. taiwanensis. Our results highlight the potential of experimental evolution in improving symbiotic proficiency and for the elucidation of relationship between symbiotic capacities and elicitation of immune responses.

The Xanthomonas type III effector XopD targets the Arabidopsis transcription factor MYB30 to suppress plant defense.

Plant and animal pathogens inject type III effectors (T3Es) into host cells to suppress host immunity and promote successful infection. XopD, a T3E from Xanthomonas campestris pv vesicatoria, has been proposed to promote bacterial growth by targeting plant transcription factors and/or regulators. Here, we show that XopD from the B100 strain of X. campestris pv campestris is able to target MYB30, a transcription factor that positively regulates Arabidopsis thaliana defense and associated cell death responses to bacteria through transcriptional activation of genes related to very-long-chain fatty acid (VLCFA) metabolism. XopD specifically interacts with MYB30, resulting in inhibition of the transcriptional activation of MYB30 VLCFA-related target genes and suppression of Arabidopsis defense. The helix-loop-helix domain of XopD is necessary and sufficient to mediate these effects. These results illustrate an original strategy developed by Xanthomonas to subvert plant defense and promote development of disease.

Bacterial host adaptation through sequence and structural variations of a single type III effector gene.

Molecular mechanisms underlying quantitative variations of pathogenicity remain elusive. Here, we identified the Xanthomonas campestris XopJ6 effector that triggers disease resistance in cauliflower and Arabidopsis thaliana. XopJ6 is a close homolog of the Ralstoniapseudosolanacearum PopP2 YopJ family acetyltransferase. XopJ6 is recognized by the RRS1-R/RPS4 NLR pair that integrates a WRKY decoy domain mimicking effector targets. We identified a XopJ6 natural variant carrying a single residue substitution in XopJ6 WRKY-binding site that disrupts interaction with WRKY proteins. This mutation allows XopJ6 to evade immune perception while retaining some XopJ6 virulence functions. Interestingly, xopJ6 resides in a Tn3-family transposon likely contributing to xopJ6 copy number variation (CNV). Using synthetic biology, we demonstrate that xopJ6 CNV tunes pathogen virulence on Arabidopsis through gene dosage-mediated modulation of xopJ6 expression. Together, our findings highlight how sequence and structural genetic variations restricted at a particular effector gene contribute to bacterial host adaptation.

AtsPLA2-alpha nuclear relocalization by the Arabidopsis transcription factor AtMYB30 leads to repression of the plant defense response.

The hypersensitive response (HR), characterized by a rapid and localized cell death at the inoculation site, is one of the most efficient resistance reactions to pathogen attack in plants. The transcription factor AtMYB30 was identified as a positive regulator of the HR and resistance responses during interactions between Arabidopsis and bacteria. Here, we show that AtMYB30 and the secreted phospholipase AtsPLA(2)-alpha physically interact in vivo, following the AtMYB30-mediated specific relocalization of AtsPLA(2)-alpha from cytoplasmic vesicles to the plant cell nucleus. This protein interaction leads to repression of AtMYB30 transcriptional activity and negative regulation of plant HR. Moreover, Atspla(2)-alpha mutant plants are more resistant to bacterial inoculation, whereas AtsPLA(2)-alpha overexpression leads to decreased resistance, confirming that AtsPLA(2)-alpha is a negative regulator of AtMYB30-mediated defense. These data underline the importance of cellular dynamics and, particularly, protein translocation to the nucleus, for defense-associated gene regulation in plants.

An efficient protocol for isolating melanised chaetothyrialean anamorphic fungi associated with plant-ants.

Because of their ecological characteristics, slow growth rates and the presence of contaminants, Chaetothyriales fungi associated with structures built by tropical plant-ants can be difficult to isolate with standard procedures. Here, we describe an easy-to-use protocol for obtaining pure cultures by using cotton as a first substrate. We have further found by means of fluorescent stains that nuclei concentrate either in young hyphae or in the tips of the hyphae.