TAL effector science

(via T. Lahaye, thx)

Hu et al, 2016

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a devastating disease of most commercial citrus varieties. In our previous study, we analyzed the transcriptional response of Valencia sweet orange to Xcc wild type and pthA4 mutant infection at 48 hours post inoculation (hpi). Using microarray analysis, two PthA4 targets, CsLOB1 and CsSWEET1, were identified. We have shown that PthA4 binds to the EBE (Effector Binding Element) of CsLOB1 and activates gene expression of this susceptibility gene. However, how PthA4 modulates host genes at different stages of infection remains to be determined. In this study, we compared the transcriptional profiles between citrus leaf tissue inoculated with Xcc306 and those inoculated with a pthA4-deletion mutant strain Xcc306∆pthA4 at 6, 48 and 120 hpi. At both 48 and 120 hpi, the PthA4-mediated infection significantly up-regulated expression of a variety of genes involved in cell wall degradation and modification, DNA packaging, G-protein, protein synthesis, sucrose metabolism and cell division functions, while the down-regulated genes were mainly enriched in photosynthesis, transport, secondary metabolism, cytochrome P450 and various plant defense associated mechanisms. To validate microarray results, gene expression of 26 genes representing genes associated with cell wall-associated, immunity system, and carbohydrate metabolism was confirmed using quantitative reverse transcription-PCR. Expression patterns of these genes at 48 and 120 hpi were consistent with the microarray results. We also identified putative Effector Binding Elements for PthA4 (EBEPthA4) in the promoter regions of multiple genes up-regulated by PthA4, to which PthA4 might bind directly to control their gene expression. Our study provided a dynamic picture of citrus genes regulated by PthA4 during the Xcc infection of citrus leaves at different stages. This study will be useful in further understanding the virulence mechanism of Xcc and identifying potential targets of PthA4.

(via T. Schreiber, thx)

Peng et al, 2016

The species Xanthomonas translucens encompasses a complex of bacterial strains that cause diseases and yield loss on grass species including important cereal crops. Three pathovars, X. translucens pv. undulosa, X. translucens pv. translucens and X. translucens pv.cerealis, have been described as pathogens of wheat, barley, and oats. However, no complete genome sequence for a strain of this complex is currently available.
A complete genome sequence of X. translucens pv. undulosa strain XT4699 was obtained by using PacBio long read, single molecule, real time (SMRT) DNA sequences and Illumina sequences. Draft genome sequences of nineteen additional X. translucens strains, which were collected from wheat or barley in different regions and at different times, were generated by Illumina sequencing. Phylogenetic relationships among different Xanthomonas strains indicates that X. translucens are members of a distinct clade from so-called group 2 xanthomonads and three pathovars of this species, undulosa, translucens and cerealis, represent distinct subclades in the group 1 clade. Knockout mutation of type III secretion system of XT4699 eliminated the ability to cause water-soaking symptoms on wheat and barley and resulted in a reduction in populations on wheat in comparison to the wild type strain. Sequence comparison of X. translucens strains revealed the genetic variation on type III effector repertories among different pathovars or within one pathovar. The full genome sequence of XT4699 reveals the presence of eight members of the Transcription-Activator Like (TAL) effector genes, which are phylogenetically distant from previous known TAL effector genes of group 2 xanthomonads. Microarray and qRT-PCR analyses revealed TAL effector-specific wheat gene expression modulation.

PacBio long read sequencing facilitates the assembly of Xanthomonas genomes and the multiple TAL effector genes, which are difficult to assemble from short read platforms. The complete genome sequence of X. translucens pv. undulosa strain XT4699 and draft genome sequences of nineteen additional X. translucens strains provides a resource for further genetic analyses of pathogenic diversity and host range of the X. translucens species complex. TAL effectors of XT4699 strain play roles in modulating wheat host gene expressions.

Schaart et al, 2015

Various new plant breeding techniques (NPBT) have a similar aim, namely to produce improved crop varieties that are difficult to obtain through traditional breeding methods. Here, we review the opportunities for products created using NPBTs. We categorize products of these NPBTs into three product classes with a different degree of genetic modification. For each product class, recent examples are described to illustrate the potential for breeding new crops with improved traits. Finally, we touch upon the future applications of these methods, such as cisgenic potato genotypes in which specific combinations of Phytophthora infestans resistance genes have been stacked for use in durable cultivation, or the creation of new disease resistances by knocking out or removing S-genes using genome-editing techniques.

Sertori et al, 2015

Zebrafish is a powerful model for the study of vertebrate development, being amenable to a wide range of genetic and other manipulations to probe the molecular basis of development and its perturbation in disease. Over recent years, genome editing approaches have become increasingly used as an efficient and sophisticated approach to precisely engineer the zebrafish genome, which has further enhanced the utility of this organism. This review provides a practical overview of genome editing and its application in zebrafish research, including alternate strategies for introducing and screening for specific genetic changes.

Cardi 2016

Plant genetic resources (PGR) represent valuable sources of genetic variability for crop breeding. The development of novel biotechnologies is necessary for increasing the efficiency of their use in pre-breeding and breeding work. The genome sequencing of hundreds of genotypes and the mining of allele diversity in major crops and populations of landraces and wild relatives allow the isolation of genes underlying characters of interest and their precise modification or transfer into targeted varieties. The technological developments and applications of new plant breeding techniques (NPBT) that maximize the similarity with gene transfer by crossing (cisgenesis/intragenesis) or the accuracy of biotechnological approaches (genome editing) are reviewed. Their potentialities and current limitations as well as the possible advantages of using them separately or combined for the exploitation of PGR in crop breeding are also discussed. Above-mentioned NPBT tackle some objections to the application of biotechnologies in agriculture and are under review worldwide to assess the possible exclusion from the current re

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Hummel et al, 2016

Xanthomonas spp. reduce crop yields and quality worldwide. During infection of their plant hosts, many strains secrete transcription activator-like (TAL) effectors, which enter the host cell nucleus and activate specific corresponding host genes at effector binding elements (EBEs) in the promoter. TAL effectors may contribute to disease by activating expression of susceptibility genes or trigger resistance associated with the hypersensitive reaction (HR) by activating an executor resistance (R) gene. The rice bacterial leaf streak pathogen X. oryzae pv. oryzicola (Xoc) is known to suppress host resistance, and no host R gene has been identified against it, despite considerable effort. To further investigate Xoc suppression of host resistance, we conducted a screen of effectors from BLS256 and identified Tal2a as an HR elicitor in rice when delivered heterologously by a strain of the closely related rice bacterial blight pathogen X. oryzae pv. oryzae (Xoo) or by the soybean pathogen X. axonopodis pv. glycines. The HR required the Tal2a activation domain, suggesting an executor R gene. Tal2a activity was differentially distributed among geographically diverse Xoc isolates, being largely conserved among Asian isolates. We identified four genes induced by Tal2a in next generation RNA sequencing experiments and confirmed them with quantitative real time RT-PCR. However, neither individual nor collective activation of these genes by designer TAL effectors resulted in the HR. A tal2a knockout mutant of BLS256 showed virulence comparable to wild type, but plasmid-based overexpression of tal2a at different levels in the wild type reduced virulence in a directly corresponding way. The results overall reveal that host resistance suppression by Xoc plays a critical role in pathogenesis. Further, the dose-dependent avirulence activity of Tal2a and the apparent lack of a single, canonical target that accounts for the HR point to a novel, activation-domain-dependent mode of action, which might involve, for example, a non-coding gene or a specific pattern of activation across multiple targets. This article is protected by copyright. All rights reserved.

Zhou et al, 2016

Programmable DNA nucleases such as TALENs and CRISPR/Cas9 are emerging as powerful tools for genome editing. Dual-fluorescent surrogate systems have been demonstrated by several studies to recapitulate DNA nuclease activity and enrich for genetically edited cells. In this study, we created a single-strand annealing-directed, dual-fluorescent surrogate reporter system, referred to as C-Check. We opted for the Golden Gate Cloning strategy to simplify C-Check construction. To demonstrate the utility of the C-Check system, we used the C-Check in combination with TALENs or CRISPR/Cas9 in different scenarios of gene editing experiments. First, we disrupted the endogenous pIAPP gene (3.0 % efficiency) by C-Check-validated TALENs in primary porcine fibroblasts (PPFs). Next, we achieved gene-editing efficiencies of 9.0–20.3 and 4.9 % when performing single- and double-gene targeting (MAPT and SORL1), respectively, in PPFs using C-Check-validated CRISPR/Cas9 vectors. Third, fluorescent tagging of endogenous genes (MYH6 and COL2A1, up to 10.0 % frequency) was achieved in human fibroblasts with C-Check-validated CRISPR/Cas9 vectors. We further demonstrated that the C-Check system could be applied to enrich for IGF1R null HEK293T cells and CBX5 null MCF-7 cells with frequencies of nearly 100.0 and 86.9 %, respectively. Most importantly, we further showed that the C-Check system is compatible with multiplexing and for studying CRISPR/Cas9 sgRNA specificity. The C-Check system may serve as an alternative dual-fluorescent surrogate tool for measuring DNA nuclease activity and enrichment of gene-edited cells, and may thereby aid in streamlining programmable DNA nuclease-mediated genome editing and biological research.

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Nishizawa-Yokoi et al, 2015

We have established methods for site-directed mutagenesis via TAL effector nucleases (TALENs) in the endogenous rice waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical non-homologous end joining (cNHEJ) and alternative-NHEJ (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA ligase4 (Lig4) affects the kinetics of TALEN-induced DSB repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations-namely deletion, insertion, combination of insertion with deletion, and substitution-in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or wild-type. In addition, the ratio of large deletions (>10-bp) and deletions repaired by micro-homology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild-type. Furthermore, almost all insertions (>= 2-bp) were shown to be processed via copy and paste of one or more regions around the TALEN-cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand-annealing.