The phytase RipBL1 enables the assignment of a specific inositol phosphate isomer as a structural component of human kidney stones.

Inositol phosphates (InsPs) are ubiquitous in all eukaryotes. However, since there are 63 possible different phosphate ester isomers, the analysis of InsPs is challenging. In particular, InsP1, InsP2, and InsP3 already amass 41 different isomers, of which some occur as enantiomers. Profiling of these "lower" inositol phosphates in mammalian tissues requires powerful analytical methods and reference compounds. Here, we report an analysis of InsP2 and InsP3 with capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS). Using this method, the bacterial effector RipBL1 was analyzed and found to degrade InsP6 to Ins(1,2,3)P3, an understudied InsP3 isomer. This new reference molecule then aided us in the assignment of the isomeric identity of an InsP3 while profiling human samples: in urine and kidney stones, we describe for the first time the presence of defined and abundant InsP3 isomers, namely Ins(1,2,3)P3, Ins(1,2,6)P3 and/or Ins(2,3,4)P3.

The tomato resistance gene Bs4 suppresses leaf watersoaking phenotypes induced by AvrHah1, a transcription activator-like effector from tomato-pathogenic xanthomonads.

The Xanthomonas transcription activator-like effector (TALE) protein AvrBs3 transcriptionally activates the executor-type resistance (R) gene Bs3 from pepper (Capsicum annuum), thereby triggering a hypersensitive cell death reaction (HR). AvrBs3 also triggers an HR in tomato (Solanum lycopersicum) upon recognition by the nucleotide-binding leucine-rich repeat (NLR) R protein Bs4. Whether the executor-type R protein Bs3 and the NLR-type R protein Bs4 use common or distinct signalling components to trigger an HR remains unclear. CRISPR/Cas9-mutagenesis revealed, that the immune signalling node EDS1 is required for Bs4- but not for Bs3-dependent HR, suggesting that NLR- and executor-type R proteins trigger an HR via distinct signalling pathways. CRISPR/Cas9-mutagenesis also revealed that tomato Bs4 suppresses the virulence function of both TALEs, the HR-inducing AvrBs3 protein and of AvrHah1, a TALE that does not trigger an HR in tomato. Analysis of AvrBs3- and AvrHah1-induced host transcripts and disease phenotypes in CRISPR/Cas9-induced bs4 mutant plants indicates that both TALEs target orthologous transcription factor genes to promote disease in tomato and pepper host plants. Our studies display that tomato mutants lacking the TALE-sensing Bs4 protein provide a novel platform to either uncover TALE-induced disease phenotypes or genetically dissect components of executor-triggered HR.

Host-associated microbe PCR (hamPCR) enables convenient measurement of both microbial load and community composition.

The ratio of microbial population size relative to the amount of host tissue, or 'microbial load', is a fundamental metric of colonization and infection, but it cannot be directly deduced from microbial amplicon data such as 16S rRNA gene counts. Because existing methods to determine load, such as serial dilution plating, quantitative PCR, and whole metagenome sequencing add substantial cost and/or experimental burden, they are only rarely paired with amplicon sequencing. We introduce host-associated microbe PCR (hamPCR), a robust strategy to both quantify microbial load and describe interkingdom microbial community composition in a single amplicon library. We demonstrate its accuracy across multiple study systems, including nematodes and major crops, and further present a cost-saving technique to reduce host overrepresentation in the library prior to sequencing. Because hamPCR provides an accessible experimental solution to the well-known limitations and statistical challenges of compositional data, it has far-reaching potential in culture-independent microbiology.

Pacbio sequencing of copper-tolerant Xanthomonas citri reveals presence of a chimeric plasmid structure and provides insights into reassortment and shuffling of transcription activator-like effectors among X. citri strains.

BACKGROUND: Xanthomonas citri, a causal agent of citrus canker, has been a well-studied model system due to recent availability of whole genome sequences of multiple strains from different geographical regions. Major limitations in our understanding of the evolution of pathogenicity factors in X. citri strains sequenced by short-read sequencing methods have been tracking plasmid reshuffling among strains due to inability to accurately assign reads to plasmids, and analyzing repeat regions among strains. X. citri harbors major pathogenicity determinants, including variable DNA-binding repeat region containing Transcription Activator-like Effectors (TALEs) on plasmids. The long-read sequencing method, PacBio, has allowed the ability to obtain complete and accurate sequences of TALEs in xanthomonads. We recently sequenced Xanthomonas citri str. Xc-03-1638-1-1, a copper tolerant A group strain isolated from grapefruit in 2003 from Argentina using PacBio RS II chemistry. We analyzed plasmid profiles, copy number and location of TALEs in complete genome sequences of X. citri strains. RESULTS: We utilized the power of long reads obtained by PacBio sequencing to enable assembly of a complete genome sequence of strain Xc-03-1638-1-1, including sequences of two plasmids, 249 kb (plasmid harboring copper resistance genes) and 99 kb (pathogenicity plasmid containing TALEs). The pathogenicity plasmid in this strain is a hybrid plasmid containing four TALEs. Due to the intriguing nature of this pathogenicity plasmid with Tn3-like transposon association, repetitive elements and multiple putative sites for origins of replication, we might expect alternative structures of this plasmid in nature, illustrating the strong adaptive potential of X. citri strains. Analysis of the pathogenicity plasmid among completely sequenced X. citri strains, coupled with Southern hybridization of the pathogenicity plasmids, revealed clues to rearrangements of plasmids and resulting reshuffling of TALEs among strains. CONCLUSIONS: We demonstrate in this study the importance of long-read sequencing for obtaining intact sequences of TALEs and plasmids, as well as for identifying rearrangement events including plasmid reshuffling. Rearrangement events, such as the hybrid plasmid in this case, could be a frequent phenomenon in the evolution of X. citri strains, although so far it is undetected due to the inability to obtain complete plasmid sequences with short-read sequencing methods.

Breaking the DNA-binding code of Ralstonia solanacearum TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease.

Ralstonia solanacearum is a devastating bacterial phytopathogen with a broad host range. Ralstonia solanacearum injected effector proteins (Rips) are key to the successful invasion of host plants. We have characterized Brg11(hrpB-regulated 11), the first identified member of a class of Rips with high sequence similarity to the transcription activator-like (TAL) effectors of Xanthomonas spp., collectively termed RipTALs. Fluorescence microscopy of in planta expressed RipTALs showed nuclear localization. Domain swaps between Brg11 and Xanthomonas TAL effector (TALE) AvrBs3 (avirulence protein triggering Bs3 resistance) showed the functional interchangeability of DNA-binding and transcriptional activation domains. PCR was used to determine the sequence of brg11 homologs from strains infecting phylogenetically diverse host plants. Brg11 localizes to the nucleus and activates promoters containing a matching effector-binding element (EBE). Brg11 and homologs preferentially activate promoters containing EBEs with a 5' terminal guanine, contrasting with the TALE preference for a 5' thymine. Brg11 and other RipTALs probably promote disease through the transcriptional activation of host genes. Brg11 and the majority of homologs identified in this study were shown to activate similar or identical target sequences, in contrast to TALEs, which generally show highly diverse target preferences. This information provides new options for the engineering of plants resistant to R. solanacearum.

RNA-seq pinpoints a Xanthomonas TAL-effector activated resistance gene in a large-crop genome.

Transcription activator-like effector (TALE) proteins of the plant pathogenic bacterial genus Xanthomonas bind to and transcriptionally activate host susceptibility genes, promoting disease. Plant immune systems have taken advantage of this mechanism by evolving TALE binding sites upstream of resistance (R) genes. For example, the pepper Bs3 and rice Xa27 genes are hypersensitive reaction plant R genes that are transcriptionally activated by corresponding TALEs. Both R genes have a hallmark expression pattern in which their transcripts are detectable only in the presence and not the absence of the corresponding TALE. By transcriptome profiling using next-generation sequencing (RNA-seq), we tested whether we could avoid laborious positional cloning for the isolation of TALE-induced R genes. In a proof-of-principle experiment, RNA-seq was used to identify a candidate for Bs4C, an R gene from pepper that mediates recognition of the Xanthomonas TALE protein AvrBs4. We identified one major Bs4C candidate transcript by RNA-seq that was expressed exclusively in the presence of AvrBs4. Complementation studies confirmed that the candidate corresponds to the Bs4C gene and that an AvrBs4 binding site in the Bs4C promoter directs its transcriptional activation. Comparison of Bs4C with a nonfunctional allele that is unable to recognize AvrBs4 revealed a 2-bp polymorphism within the TALE binding site of the Bs4C promoter. Bs4C encodes a structurally unique R protein and Bs4C-like genes that are present in many solanaceous genomes seem to be as tightly regulated as pepper Bs4C. These findings demonstrate that TALE-specific R genes can be cloned from large-genome crops with a highly efficient RNA-seq approach.