scholarly journals I-SceI Endonuclease-Mediated Plant Genome Editing by Protein Transport through a Bacterial Type III Secretion System

Plants ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1070
Author(s):  
Yuki Yanagawa ◽  
Kasumi Takeuchi ◽  
Masaki Endo ◽  
Ayako Furutani ◽  
Hirokazu Ochiai ◽  
...  
Keyword(s):  
Genome Editing ◽  
Type Iii Secretion ◽  
Tobacco Plant ◽  
Plant Cells ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Plant Genome ◽  
Recognition Sequence ◽  
Type Iii ◽  
A Genome

Xanthomonas campestris is one of bacteria carrying a type III secretion system which transports their effector proteins into host plant cells to disturb host defense system for their infection. To establish a genome editing system without introducing any foreign gene, we attempted to introduce genome editing enzymes through the type III secretion system. In a test of protein transfer, X. campestris pv. campestris (Xcc) transported a considerable amount of a reporter protein sGFP-CyaA into tobacco plant cells under the control of the type III secretion system while maintaining cell viability. For proof of concept for genome editing, we used a reporter tobacco plant containing a luciferase (LUC) gene interrupted by a meganuclease I-SceI recognition sequence; this plant exhibits chemiluminescence of LUC only when a frameshift mutation is introduced at the I-SceI recognition site. Luciferase signal was observed in tobacco leaves infected by Xcc carrying an I-SceI gene which secretes I-SceI protein through the type III system, but not leaves infected by Xcc carrying a vector control. Genome-edited tobacco plant could be regenerated from a piece of infected leaf piece by repeated selection of LUC positive calli. Sequence analysis revealed that the regenerated tobacco plant possessed a base deletion in the I-SceI recognition sequence that activated the LUC gene, indicating genome editing by I-SceI protein transferred through the type III secretion system of Xcc.

scholarly journals Pseudomonas syringae Strains Naturally Lacking the Classical P. syringae hrp/hrc Locus Are Common Leaf Colonizers Equipped with an Atypical Type III Secretion System

2010 ◽  
Vol 23 (2) ◽  
pp. 198-210 ◽  
Author(s):  
Christopher R. Clarke ◽  
Rongman Cai ◽  
David J. Studholme ◽  
David S. Guttman ◽  
Boris A. Vinatzer
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Plant Cells ◽  
Type Iii Secretion System ◽  
Ice Nucleation ◽  
Secretion System ◽  
Effector Proteins ◽  
Population Densities ◽  
Type Iii ◽  
Effector Genes

Pseudomonas syringae is best known as a plant pathogen that causes disease by translocating immune-suppressing effector proteins into plant cells through a type III secretion system (T3SS). However, P. syringae strains belonging to a newly described phylogenetic subgroup (group 2c) are missing the canonical P. syringae hrp/hrc cluster coding for a T3SS, flanking effector loci, and any close orthologue of known P. syringae effectors. Nonetheless, P. syringae group 2c strains are common leaf colonizers and grow on some tested plant species to population densities higher than those obtained by other P. syringae strains on nonhost species. Moreover, group 2c strains have genes necessary for the production of phytotoxins, have an ice nucleation gene, and, most interestingly, contain a novel hrp/hrc cluster, which is only distantly related to the canonical P. syringae hrp/hrc cluster. This hrp/hrc cluster appears to encode a functional T3SS although the genes hrpK and hrpS, present in the classical P. syringae hrp/hrc cluster, are missing. The genome sequence of a representative group 2c strain also revealed distant orthologues of the P. syringae effector genes avrE1 and hopM1 and the P. aeruginosa effector genes exoU and exoY. A putative life cycle for group 2c P. syringae is discussed.

scholarly journals Identification of Pseudomonas syringae pv. syringae 61 Type III Secretion System Hrp Proteins That Can Travel the Type III Pathway and Contribute to the Translocation of Effector Proteins into Plant Cells

2007 ◽  
Vol 189 (15) ◽  
pp. 5773-5778 ◽  
Author(s):  
Adela R. Ramos ◽  
Joanne E. Morello ◽  
Sandeep Ravindran ◽  
Wen-Ling Deng ◽  
Hsiou-Chen Huang ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Hypersensitive Response ◽  
Type Iii Secretion ◽  
Plant Cells ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Type Iii

ABSTRACT Pseudomonas syringae translocates effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). T3SS components HrpB, HrpD, HrpF, and HrpP were shown to be pathway substrates and to contribute to elicitation of the plant hypersensitive response and to translocation and secretion of the model effector AvrPto1.

scholarly journals Pseudomonas syringae Type III Secretion System Targeting Signals and Novel Effectors Studied with a Cya Translocation Reporter

2004 ◽  
Vol 186 (2) ◽  
pp. 543-555 ◽  
Author(s):  
Lisa M. Schechter ◽  
Kathy A. Roberts ◽  
Yashitola Jamir ◽  
James R. Alfano ◽  
Alan Collmer
Keyword(s):  
Amino Acids ◽  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Plant Cells ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Reporter System ◽  
Aliphatic Amino Acid ◽  
Type Iii

ABSTRACT Pseudomonas syringae pv. tomato strain DC3000 is a pathogen of tomato and Arabidopsis. The hrp-hrc-encoded type III secretion system (TTSS), which injects bacterial effector proteins (primarily called Hop or Avr proteins) into plant cells, is required for pathogenicity. In addition to being regulated by the HrpL alternative sigma factor, most avr or hop genes encode proteins with N termini that have several characteristic features, including (i) a high percentage of Ser residues, (ii) an aliphatic amino acid (Ile, Leu, or Val) or Pro at the third or fourth position, and (iii) a lack of negatively charged amino acids within the first 12 residues. Here, the well-studied effector AvrPto was used to optimize a calmodulin-dependent adenylate cyclase (Cya) reporter system for Hrp-mediated translocation of P. syringae TTSS effectors into plant cells. This system includes a cloned P. syringae hrp gene cluster and the model plant Nicotiana benthamiana. Analyses of truncated AvrPto proteins fused to Cya revealed that the N-terminal 16 amino acids and/or codons of AvrPto are sufficient to direct weak translocation into plant cells and that longer N-terminal fragments direct progressively stronger translocation. AvrB, tested because it is poorly secreted in cultures by the P. syringae Hrp system, was translocated into plant cells as effectively as AvrPto. The translocation of several DC3000 candidate Hop proteins was also examined by using Cya as a reporter, which led to identification of three new intact Hop proteins, designated HopPtoQ, HopPtoT1, and HopPtoV, as well as two truncated Hop proteins encoded by the naturally disrupted genes hopPtoS4::tnpA and hopPtoAG::tnpA. We also confirmed that HopPtoK, HopPtoC, and AvrPphEPto are translocated into plant cells. These results increased the number of Hrp system-secreted proteins in DC3000 to 40. Although most of the newly identified Hop proteins possess N termini that have the same features as the N termini of previously described Hop proteins, HopPtoV has none of these characteristics. Our results indicate that Cya should be a useful reporter for exploring multiple aspects of the Hrp system in P. syringae.

scholarly journals Naturally Occurring Nonpathogenic Isolates of the Plant Pathogen Pseudomonas syringae Lack a Type III Secretion System and Effector Gene Orthologues

2008 ◽  
Vol 190 (8) ◽  
pp. 2858-2870 ◽  
Author(s):  
Toni J. Mohr ◽  
Haijie Liu ◽  
Shuangchun Yan ◽  
Cindy E. Morris ◽  
José A. Castillo ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Plant Cells ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Plant Diseases ◽  
In Planta ◽  
Type Iii ◽  
Effector Gene

ABSTRACT Pseudomonas syringae causes plant diseases, and the main virulence mechanism is a type III secretion system (T3SS) that translocates dozens of effector proteins into plant cells. Here we report the existence of a subgroup of P. syringae isolates that do not cause disease on any plant species tested. This group is monophyletic and most likely evolved from a pathogenic P. syringae ancestor through loss of the T3SS. In the nonpathogenic isolate P. syringae 508 the genomic region that in pathogenic P. syringae strains contains the hrp-hrc cluster coding for the T3SS and flanking effector genes is absent. P. syringae 508 was also surveyed for the presence of effector orthologues from the closely related pathogenic strain P. syringae pv. syringae B728a, but none were detected. The absence of the hrp-hrc cluster and effector orthologues was confirmed for other nonpathogenic isolates. Using the AvrRpt2 effector as reporter revealed the inability of P. syringae 508 to translocate effectors into plant cells. Adding a plasmid-encoded T3SS and the P. syringae pv. syringae 61 effector gene hopA1 increased in planta growth almost 10-fold. This suggests that P. syringae 508 supplemented with a T3SS could be used to determine functions of individual effectors in the context of a plant infection, avoiding the confounding effect of other effectors with similar functions present in effector mutants of pathogenic isolates.

scholarly journals Pseudomonas syringae Lytic Transglycosylases Coregulated with the Type III Secretion System Contribute to the Translocation of Effector Proteins into Plant Cells

2007 ◽  
Vol 189 (22) ◽  
pp. 8277-8289 ◽  
Author(s):  
Hye-Sook Oh ◽  
Brian H. Kvitko ◽  
Joanne E. Morello ◽  
Alan Collmer
Keyword(s):  
Pseudomonas Syringae ◽  
Hypersensitive Response ◽  
Type Iii Secretion ◽  
Plant Cells ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Effector Proteins ◽  
Dominant Negative Effect ◽  
Lytic Transglycosylases ◽  
Type Iii

ABSTRACT Pseudomonas syringae translocates virulence effector proteins into plant cells via a type III secretion system (T3SS) encoded by hrp (for hypersensitive response and pathogenicity) genes. Three genes coregulated with the Hrp T3SS system in P. syringae pv. tomato DC3000 have predicted lytic transglycosylase domains: PSPTO1378 (here designated hrpH), PSPTO2678 (hopP1), and PSPTO852 (hopAJ1). hrpH is located between hrpR and avrE1 in the Hrp pathogenicity island and is carried in the functional cluster of P. syringae pv. syringae 61 hrp genes cloned in cosmid pHIR11. Strong expression of DC3000 hrpH in Escherichia coli inhibits bacterial growth unless the predicted catalytic glutamate at position 148 is mutated. Translocation tests involving C-terminal fusions with a Cya (Bordetella pertussis adenylate cyclase) reporter indicate that HrpH and HopP1, but not HopAJ1, are T3SS substrates. Pseudomonas fluorescens carrying a pHIR11 derivative lacking hrpH is poorly able to translocate effector HopA1, and this deficiency can be restored by HopP1 and HopAJ1, but not by HrpH(E148A) or HrpH1-241. DC3000 mutants lacking hrpH or hrpH, hopP1, and hopAJ1 combined are variously reduced in effector translocation, elicitation of the hypersensitive response, and virulence. However, the mutants are not reduced in secretion of T3SS substrates in culture. When produced in wild-type DC3000, the HrpH(E148A) and HrpH1-241 variants have a dominant-negative effect on the ability of DC3000 to elicit the hypersensitive response in nonhost tobacco and to grow and cause disease in host tomato. The three Hrp-associated lytic transglycosylases in DC3000 appear to have overlapping functions in contributing to T3SS functions during infection.

Sign in / Sign up

Export Citation Format

Share Document