Prevalence and diversity of TAL effector-like proteins in fungal endosymbiotic Mycetohabitans spp.

Endofungal Mycetohabitans (formerly Burkholderia) spp. rely on a type III secretion system to deliver mostly unidentified effector proteins when colonizing their host fungus, Rhizopus microsporus. The one known secreted effector family from Mycetohabitans consists of homologues of transcription activator-like (TAL) effectors, which are used by plant pathogenic Xanthomonas and Ralstonia spp. to activate host genes that promote disease. These 'Burkholderia TAL-like (Btl)' proteins bind corresponding specific DNA sequences in a predictable manner, but their genomic target(s) and impact on transcription in the fungus are unknown. Recent phenotyping of Btl mutants of two Mycetohabitans strains revealed that the single Btl in one Mycetohabitans endofungorum strain enhances fungal membrane stress tolerance, while others in a Mycetohabitans rhizoxinica strain promote bacterial colonization of the fungus. The phenotypic diversity underscores the need to assess the sequence diversity and, given that sequence diversity translates to DNA targeting specificity, the functional diversity of Btl proteins. Using a dual approach to maximize capture of Btl protein sequences for our analysis, we sequenced and assembled nine Mycetohabitans spp. genomes using long-read PacBio technology and also mined available short-read Illumina fungal-bacterial metagenomes. We show that btl genes are present across diverse Mycetohabitans strains from Mucoromycota fungal hosts yet vary in sequences and predicted DNA binding specificity. Phylogenetic analysis revealed distinct clades of Btl proteins and suggested that Mycetohabitans might contain more species than previously recognized. Within our data set, Btl proteins were more conserved across M. rhizoxinica strains than across M. endofungorum, but there was also evidence of greater overall strain diversity within the latter clade. Overall, the results suggest that Btl proteins contribute to bacterial-fungal symbioses in myriad ways.

Cruciferous Weed Isolates of Xanthomonas campestris Yield Insight into Pathovar Genomic Relationships and Genetic Determinants of Host and Tissue Specificity.

Pathovars of Xanthomonas campestris cause distinct diseases on different brassicaceous hosts. The genomic relationships among pathovars as well as the genetic determinants of host range and tissue specificity remain poorly understood despite decades of research. Here, leveraging advances in multiplexed long-read technology, we fully sequenced the genomes of a collection of X. campestris strains isolated from cruciferous crops and weeds in New York and California as well as strains from global collections, to investigate pathovar relationships and candidate genes for host- and tissue-specificity. Pathogenicity assays and genomic comparisons across this collection and publicly available X. campestris genomes revealed a correlation between pathovar and genomic relatedness and provide support for X. campestris pv. barbareae, the validity of which had been questioned. Linking strain host range with type III effector repertoires identified AvrAC (also 'XopAC') as a candidate host-range determinant, preventing infection of Matthiola incana, and this was confirmed experimentally. Furthermore, the presence of a copy of the cellobiosidase gene cbsA with coding sequence for a signal peptide was found to correlate with the ability to infect vascular tissues, in agreement with a previous study of diverse Xanthomonas species; however, heterologous expression in strains lacking the gene gave mixed results, indicating that factors in addition to cbsA influence tissue specificity of X. campestris pathovars. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Transcriptional Profiles of a Foliar Fungal Endophyte (Pestalotiopsis, Ascomycota) and Its Bacterial Symbiont (Luteibacter, Gammaproteobacteria) Reveal Sulfur Exchange and Growth Regulation during Early Phases of Symbiotic Interaction.

Symbiosis with bacteria is widespread among eukaryotes, including fungi. Bacteria that live within fungal mycelia (endohyphal bacteria) occur in many plant-associated fungi, including diverse Mucoromycota and Dikarya. Pestalotiopsis sp. strain 9143 is a filamentous ascomycete isolated originally as a foliar endophyte of Platycladus orientalis (Cupressaceae). It is infected naturally with the endohyphal bacterium Luteibacter sp. strain 9143, which influences auxin and enzyme production by its fungal host. Previous studies have used transcriptomics to examine similar symbioses between endohyphal bacteria and root-associated fungi such as arbuscular mycorrhizal fungi and plant pathogens. However, currently there are no gene expression studies of endohyphal bacteria of Ascomycota, the most species-rich fungal phylum. To begin to understand such symbioses, we developed methods for assessing gene expression by Pestalotiopsis sp. and Luteibacter sp. when grown in coculture and when each was grown axenically. Our assays showed that the density of Luteibacter sp. in coculture was greater than in axenic culture, but the opposite was true for Pestalotiopsis sp. Dual-transcriptome sequencing (RNA-seq) data demonstrate that growing in coculture modulates developmental and metabolic processes in both the fungus and bacterium, potentially through changes in the balance of organic sulfur via methionine acquisition. Our analyses also suggest an unexpected, potential role of the bacterial type VI secretion system in symbiosis establishment, expanding current understanding of the scope and dynamics of fungal-bacterial symbioses. IMPORTANCE Interactions between microbes and their hosts have important outcomes for host and environmental health. Foliar fungal endophytes that infect healthy plants can harbor facultative endosymbionts called endohyphal bacteria, which can influence the outcome of plant-fungus interactions. These bacterial-fungal interactions can be influential but are poorly understood, particularly from a transcriptome perspective. Here, we report on a comparative, dual-RNA-seq study examining the gene expression patterns of a foliar fungal endophyte and a facultative endohyphal bacterium when cultured together versus separately. Our findings support a role for the fungus in providing organic sulfur to the bacterium, potentially through methionine acquisition, and the potential involvement of a bacterial type VI secretion system in symbiosis establishment. This work adds to the growing body of literature characterizing endohyphal bacterial-fungal interactions, with a focus on a model facultative bacterial-fungal symbiosis in two species-rich lineages, the Ascomycota and Proteobacteria.

A TAL effector-like protein of an endofungal bacterium increases the stress tolerance and alters the transcriptome of the host.

Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis of Mycetohabitans (formerly Burkholderia) rhizoxinica with the fungus Rhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequenced Mycetohabitans spp. genomes. TAL effectors nuclear-localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions, in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear-localize. A btl19-13 gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15 R. microsporus genes were differentially expressed in comparisons both of the fungus infected with the wild-type bacterium vs. the mutant and with the mutant vs. a complemented strain. Southern blotting revealed btl genes in 14 diverse Mycetohabitans isolates. However, banding patterns and available sequences suggest variation, and the btl19-13 phenotype could not be rescued by a btl gene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host genotype-specific roles in the M. rhizoxinica-R. microsporus symbiosis.

A polyketide synthase gene cluster associated with the sexual reproductive cycle of the banana pathogen, Pseudocercospora fijiensis.

Disease spread of Pseudocercospora fijiensis, causal agent of the black Sigatoka disease of banana, depends on ascospores produced through the sexual reproductive cycle. We used phylogenetic analysis to identify P. fijiensis homologs (PKS8-4 and Hybrid8-3) to the PKS4 polyketide synthases (PKS) from Neurospora crassa and Sordaria macrospora involved in sexual reproduction. These sequences also formed a clade with lovastatin, compactin, and betaenone-producing PKS sequences. Transcriptome analysis showed that both the P. fijiensis Hybrid8-3 and PKS8-4 genes have higher expression in infected leaf tissue compared to in culture. Domain analysis showed that PKS8-4 is more similar than Hybrid8-3 to PKS4. pPKS8-4:GFP transcriptional fusion transformants showed expression of GFP in flask-shaped structures in mycelial cultures as well as in crosses between compatible and incompatible mating types. Confocal microscopy confirmed expression in spermagonia in leaf substomatal cavities, consistent with a role in sexual reproduction. A disruption mutant of pks8-4 retained normal pathogenicity on banana, and no differences were observed in growth, conidial production, and spermagonia production. GC-MS profiling of the mutant and wild type did not identify differences in polyketide metabolites, but did identify changes in saturated fatty acid methyl esters and alkene and alkane derivatives. To our knowledge, this is the first report of a polyketide synthase pathway associated with spermagonia.

Convergent Evolution of Effector Protease Recognition by Arabidopsis and Barley.

The Pseudomonas syringae cysteine protease AvrPphB activates the Arabidopsis resistance protein RPS5 by cleaving a second host protein, PBS1. AvrPphB induces defense responses in other plant species, but the genes and mechanisms mediating AvrPphB recognition in those species have not been defined. Here, we show that AvrPphB induces defense responses in diverse barley cultivars. We also show that barley contains two PBS1 orthologs, that their products are cleaved by AvrPphB, and that the barley AvrPphB response maps to a single locus containing a nucleotide-binding leucine-rich repeat (NLR) gene, which we termed AvrPphB Response 1 (Pbr1). Transient coexpression of PBR1 with wild-type AvrPphB but not with a protease inactive mutant triggered defense responses, indicating that PBR1 detects AvrPphB protease activity. Additionally, PBR1 coimmunoprecipitated with barley and Nicotiana benthamiana PBS1 proteins, suggesting mechanistic similarity to detection by RPS5. Lastly, we determined that wheat cultivars also recognize AvrPphB protease activity and contain two putative Pbr1 orthologs. Phylogenetic analyses showed, however, that Pbr1 is not orthologous to RPS5. Our results indicate that the ability to recognize AvrPphB evolved convergently and imply that selection to guard PBS1-like proteins occurs across species. Also, these results suggest that PBS1-based decoys may be used to engineer protease effector recognition-based resistance in barley and wheat.

A Confounding Effect of Bacterial Titer in a Type III Delivery-Based Assay of Eukaryotic Effector Function.

This letter describes a newly discovered confounding effect of bacterial titer in a previously published type III delivery-based assay of the fungal effector BEC1019. The original publication (Whigham et al. 2015) has been retracted as a consequence of this discovery. Here, we tabulate the affected and unaffected figures and conclusions in the original publication and briefly reflect on potential pitfalls to bear in mind when designing experiments that use bacterial type III secretion to characterize eukaryotic effectors.