Influence of affinity tags and tobacco PR1a signal peptide on detection, purification and bioactivity analyses of the small oomycete apoplastic effectors.
Biotechnol Lett
; 45(1): 115-124, 2023 Jan.
Article
em En
| MEDLINE
| ID: mdl-36450976
ABSTRACT
OBJECTIVE:
To examine the influence of widely used protein affinity tags and the tobacco PR1a signal peptide (SP) on detection, purification and bioactivity analyses of the small oomycete apoplastic effector SCR96 in planta.RESULTS:
Through agroinfiltration, the phytotoxic effector SCR96 of Phytophthora cactorum was expressed in Nicotiana benthamiana leaf apoplast as a fusion protein carrying single affinity tag (His, HA or FLAG) at either C- or N-terminus. Leaf necrosis caused by different affinity-tagged SCR96 varied among tags and replicates. All of tagged proteins can be detected by antibodies against SCR96. All of SCR96 fusions except N-terminally fused 6His-tagged protein were detected using tag antibodies, indicating that 6His tag may be degraded when fused at N-terminus. Interestingly, C-terminal His- and FLAG-tagged SCR96 maintained the biological activity after purification. In the substitution assay of SCR96 SP, we observed that PR1a SP can lead chimeric SCR96 expression in N. benthamiana, but the replacement totally disrupted its bioactivity.CONCLUSION:
C-terminal His or FLAG tag, along with its original SP, is efficient enough to enable detection and purification of functional SCR96 from N. benthamiana leaf apoplast, which would facilitate plant-pathogen interaction studies.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Phytophthora
/
Nicotiana
Tipo de estudo:
Diagnostic_studies
Idioma:
En
Revista:
Biotechnol Lett
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
China