TAL effector
Encyclopedia
TAL effectors (often referred to as TALEs but not to be confused with the three amino acid loop extension family of proteins) are protein
s secreted by Xanthomonas
bacteria via their type III secretion system
when they infect various plant
species. These proteins can bind promoter sequences in the host plant and activate the expression
of plant genes that aid bacterial infection. They recognize plant DNA sequence
s through a central repeat domain consisting of a variable number of ~34 amino acid repeats. There appears to be a one-to-one correspondence between the identity of two critical amino acids in each repeat and each DNA base in the target sequence. These proteins are interesting to researchers both for their role in disease of important crop species and the relative ease of retargeting them to bind new DNA sequences. Similar proteins can be found in the pathogenic bacteria Ralstonia solanacearum
.
Some types of Xanthomonas cause localized leaf spot or leaf streak while others spread systemically and cause black rot or leaf blight disease. They inject a number of effector proteins, including TAL effectors, into the plant via their type III secretion system
. TAL effectors (short for Transcription Activator-like effectors) have several motifs normally associated with eukaryotes including multiple nuclear localization signals and an acidic activation domain. When injected into plants, these proteins can enter the nucleus of the plant cell, bind plant promoter sequences, and activate transcription of plant genes that aid in bacterial infection. Plants have developed a defense mechanism against type III effectors that includes R (resistance) genes triggered by these effectors. Some of these R genes appear to have evolved to contain TAL-effector binding sites similar to site in the intended target gene. This competition between pathogenic bacteria and the host plant has been hypothesized to account for the apparently malleable nature of the TAL effector DNA binding domain.
genes in tomato
, Arabidopsis thaliana
, and human cells. Engineered TAL effectors can also be fused to the cleavage domain of FokI
to create TAL effector nucleases
(TALENs). Such nucleases share some properties with zinc finger nucleases and may be useful for genetic engineering
and gene therapy
applications. TALENs show activity in a yeast-based assay, at endogenous yeast genes, in a plant reporter assay, at an endogenous plant gene, at endogenous zebrafish genes, at an endogenous rat
gene, and at endogenous human genes. The human HPRT1 gene has been targeted at detectable, but unquantified levels and TALENs containing the FokI cleavage domain fused to a smaller portion of the TAL effector still containing the DNA binding domain have been used to target the endogenous NTF3 and CCR5
genes in human cells with efficiencies of up to 25%. TAL effector nucleases have also been used to engineer human embryonic stem cell
s and induced pluripotent stem cells
(IPSCs) and to knock out the endogenous ben-1 gene in C. elegans
. Genetic constructs to encode TAL effector-based proteins can be made using either conventional gene synthesis or modular assembly. A plasmid kit for assembling custom TALEN and other TAL effector constructs is available through the public, not-for-profit repository AddGene. Webpages providing access to public software, protocols, and other resources for TAL effector-DNA targeting applications include the TAL Effector-Nucleotide Targeter and taleffectors.com. TAL effector nuclease constructs are commercially available from a European biotechnology company.
Two hypotheses exist about possible functions for N3 proteins:
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s secreted by Xanthomonas
Xanthomonas
Xanthomonas is a genus of Proteobacteria, many of which cause plant diseases. Most varieties of Xanthomonas are available from the National Collection of Plant Pathogenic Bacteria in the United Kingdom and other international culture collections such as ICMP in New Zealand, CFBP in France, and...
bacteria via their type III secretion system
Type three secretion system
Type three secretion system is a protein appendage found in several Gram-negative bacteria....
when they infect various plant
Plant
Plants are living organisms belonging to the kingdom Plantae. Precise definitions of the kingdom vary, but as the term is used here, plants include familiar organisms such as trees, flowers, herbs, bushes, grasses, vines, ferns, mosses, and green algae. The group is also called green plants or...
species. These proteins can bind promoter sequences in the host plant and activate the expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
of plant genes that aid bacterial infection. They recognize plant DNA sequence
DNA sequence
The sequence or primary structure of a nucleic acid is the composition of atoms that make up the nucleic acid and the chemical bonds that bond those atoms. Because nucleic acids, such as DNA and RNA, are unbranched polymers, this specification is equivalent to specifying the sequence of...
s through a central repeat domain consisting of a variable number of ~34 amino acid repeats. There appears to be a one-to-one correspondence between the identity of two critical amino acids in each repeat and each DNA base in the target sequence. These proteins are interesting to researchers both for their role in disease of important crop species and the relative ease of retargeting them to bind new DNA sequences. Similar proteins can be found in the pathogenic bacteria Ralstonia solanacearum
Ralstonia solanacearum
Ralstonia solanacearum is an aerobic non-sporing, Gram-negative plant pathogenic bacterium. R. solanacearum is soil-borne and motile with a polar flagellar tuft. It colonises the xylem, causing bacterial wilt in a very wide range of potential host plants...
.
Function in plant pathogenesis
Xanthomonas are gram-negative bacteria that can infect a wide variety of plant species including pepper, rice, citrus, cotton, tomato, and soybeans.Some types of Xanthomonas cause localized leaf spot or leaf streak while others spread systemically and cause black rot or leaf blight disease. They inject a number of effector proteins, including TAL effectors, into the plant via their type III secretion system
Type three secretion system
Type three secretion system is a protein appendage found in several Gram-negative bacteria....
. TAL effectors (short for Transcription Activator-like effectors) have several motifs normally associated with eukaryotes including multiple nuclear localization signals and an acidic activation domain. When injected into plants, these proteins can enter the nucleus of the plant cell, bind plant promoter sequences, and activate transcription of plant genes that aid in bacterial infection. Plants have developed a defense mechanism against type III effectors that includes R (resistance) genes triggered by these effectors. Some of these R genes appear to have evolved to contain TAL-effector binding sites similar to site in the intended target gene. This competition between pathogenic bacteria and the host plant has been hypothesized to account for the apparently malleable nature of the TAL effector DNA binding domain.
DNA recognition
The most distinctive characteristic of TAL effectors is a central repeat domain containing between 1.5 and 33.5 repeats that are usually 34 residues in length (the C-terminal repeat is generally shorter and referred to as a “half repeat”). A typical repeat sequence is LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG, but the residues at the 12th and 13th positions are hypervariable (these two amino acids are also known as the repeat variable diresidue or RVD). Two separate groups have shown that there is a simple relationship between the identity of these two residues in sequential repeats and sequential DNA bases in the TAL effector’s target site. The first group broke this code computationally by searching for patterns in protein sequence alignments and DNA sequences of target promoters. The second group deduced the code through molecular analysis of the TAL effector AvrBs3 and its target DNA sequence in the promoter of a pepper gene activated by AvrBs3. The experimentally validated code between RVD sequence and target DNA base can be expressed as NI = A, HD = C, NG = T, NN = G or A, and NS = A, C, G, or T. Further studies has shown that the RVD NK can target G. Target sites of TAL effectors also tend to include a T flanking the 5’ base targeted by the first repeat, but the exact mechanism of this recognition is not known.Engineered TAL effectors
This simple code between amino acids in TAL effectors and DNA bases in their target sites might be useful for protein engineering applications. Numerous groups have designed artificial TAL effectors capable of recognizing new DNA sequences in a variety of experimental systems. Such engineered TAL effectors have been used to create artificial transcription factors that can be used to target and activate endogenousEndogenous
Endogenous substances are those that originate from within an organism, tissue, or cell. Endogenous retroviruses are caused by ancient infections of germ cells in humans, mammals and other vertebrates...
genes in tomato
Tomato
The word "tomato" may refer to the plant or the edible, typically red, fruit which it bears. Originating in South America, the tomato was spread around the world following the Spanish colonization of the Americas, and its many varieties are now widely grown, often in greenhouses in cooler...
, Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana is a small flowering plant native to Europe, Asia, and northwestern Africa. A spring annual with a relatively short life cycle, arabidopsis is popular as a model organism in plant biology and genetics...
, and human cells. Engineered TAL effectors can also be fused to the cleavage domain of FokI
FokI
The enzyme FokI, naturally found in Flavobacterium okeanokoites, is a bacterial type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and a non-specific DNA cleavage domain at the C-terminal...
to create TAL effector nucleases
Transcription Activator-Like Effector Nuclease
Transcription Activator-Like Effector Nucleases are artificial restriction enzymes generated by fusing the TAL effector DNA binding domain to a DNA cleavage domain.-Use:...
(TALENs). Such nucleases share some properties with zinc finger nucleases and may be useful for genetic engineering
Genetic engineering
Genetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...
and gene therapy
Gene therapy
Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development...
applications. TALENs show activity in a yeast-based assay, at endogenous yeast genes, in a plant reporter assay, at an endogenous plant gene, at endogenous zebrafish genes, at an endogenous rat
Rat
Rats are various medium-sized, long-tailed rodents of the superfamily Muroidea. "True rats" are members of the genus Rattus, the most important of which to humans are the black rat, Rattus rattus, and the brown rat, Rattus norvegicus...
gene, and at endogenous human genes. The human HPRT1 gene has been targeted at detectable, but unquantified levels and TALENs containing the FokI cleavage domain fused to a smaller portion of the TAL effector still containing the DNA binding domain have been used to target the endogenous NTF3 and CCR5
CCR5
C-C chemokine receptor type 5, also known as CCR5, is a protein that in humans is encoded by the CCR5 gene. CCR5 is a member of the beta chemokine receptors family of integral membrane proteins...
genes in human cells with efficiencies of up to 25%. TAL effector nucleases have also been used to engineer human embryonic stem cell
Embryonic stem cell
Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consist of 50–150 cells...
s and induced pluripotent stem cells
Induced pluripotent stem cell
Induced pluripotent stem cells, commonly abbreviated as iPS cells or iPSCs are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, by inducing a "forced" expression of specific genes....
(IPSCs) and to knock out the endogenous ben-1 gene in C. elegans
Caenorhabditis elegans
Caenorhabditis elegans is a free-living, transparent nematode , about 1 mm in length, which lives in temperate soil environments. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner and it has since been used extensively as a model...
. Genetic constructs to encode TAL effector-based proteins can be made using either conventional gene synthesis or modular assembly. A plasmid kit for assembling custom TALEN and other TAL effector constructs is available through the public, not-for-profit repository AddGene. Webpages providing access to public software, protocols, and other resources for TAL effector-DNA targeting applications include the TAL Effector-Nucleotide Targeter and taleffectors.com. TAL effector nuclease constructs are commercially available from a European biotechnology company.
Target genes
TAL effectors can induce susceptibility genes that are members of the NODULIN3 (N3) gene family. These genes are essential for the development of the disease. In rice two genes,Os-8N3 and Os-11N3, are induced by TAL effectors. Os-8N3 is induced by PthXo1 and Os-11N3 is induced by PthXo3 and AvrXa7.Two hypotheses exist about possible functions for N3 proteins:
- They are involved in copper transport, resulting in detoxification of the environment for bacteria. The reduction in copper level facilitates bacterial growth.
- They are involved in glucose transport, facilitating glucose flow. This mechanism provides nutrients to bacteria and stimulates pathogen growth and virulence
See also
- DNA-binding proteinDNA-binding proteinDNA-binding proteins are proteins that are composed of DNA-binding domains and thus have a specific or general affinity for either single or double stranded DNA. Sequence-specific DNA-binding proteins generally interact with the major groove of B-DNA, because it exposes more functional groups that...
- DNA binding domain
- Sequence motifSequence motifIn genetics, a sequence motif is a nucleotide or amino-acid sequence pattern that is widespread and has, or is conjectured to have, a biological significance...
- Zinc fingerZinc fingerZinc fingers are small protein structural motifs that can coordinate one or more zinc ions to help stabilize their folds. They can be classified into several different structural families and typically function as interaction modules that bind DNA, RNA, proteins, or small molecules...
- Zinc finger nucleaseZinc finger nucleaseZinc-finger nucleases are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes...
External links
- Cellectis bioresearch A commercial supplier of TALENs
- www.taleffectors.com An open resource for TAL effector constructs