Quantitative phosphoproteomic analysis reveals common
1. AREA:
Interacción Molecular Planta Patogeno
DOCENTE:
-Jose M. Soto Heredia
ALUMNOS:
- Diego Fernando Luján Ajalcriña (20200601)
- Miguel Maturrano
El análisis fosfoproteómico cuantitativo revela
mecanismos reguladores comunes entre la
inmunidad activada por efectores y PAMP en plantas
4. INTRODUCCION
Sistema de secreción III (T3SS):
Existen de dos tipos:
• No-flagelar (NF)
• Flagelar (F)
Función:
Inyectar EFECTORES de tipo III
(T3Fs).
(Ji & Dong, 2015)
Componentes:
• ATPase asociado al citoplasma
(HrcQ)
• Proteína embebida en la membrana
del citoplasma
(HrcU, etc.) que forma un anillo.
• Otra proteína que forma un anillo
pero que abarca
todo el espacio periplásmico (HrcJ) que
conecta la
IM con la OM.
• Secretinas que forma un anillo o poro
(HrcC).
5.
6. OBJETIVOS
Proporcionar datos fosfoproteómicos importantes que contribuyen a la comprensión de la red de
señalización de ETI y mostrar puntos en común y diferencias con respecto a la red de señalización
de PTI.
7. MATERIALES Y METODOS
Condiciones de crecimiento :
H.R.: 70%
Temperatura: 23°C
Intensidad de luz de 85 μmol m- 2s - 1
Fotoperiodo de 10 h de luz y 14 h de
oscuridad
GVG:avrRpt2 X rpm1-3 rps2-101C
rpm1rps2/GVG:avrRpt2
(rpm1rps2/Dex:avrRpt2)
LINEAS DE COMPLEMNETACION:
Líneas de complementación etiquetadas con RbohD rbohD/35S:FLAG-RbohD
(rbohD/35S:FLAG-RbohD), rbohD/pRbohD:3xFLAG-RbohD
(rbohD/pRbohD:FLAG-RbohD) y rbohD/pRbohD:3xFLAG-RbohD-S343A/S347A
(rbohD/pRbohD:FLAG-RbohD-S343A/S347A)
3xFLAG-RbohD X rbohD/pRbohD:3xFLAG-RbohD-S343A-S347A
rbohDrbohF +
8. MATERIALES Y METODOS
Se aplicó :
Dex 30 μM y
Silwett L-77 al
0,025 %.V
Preparación de muestras de fosfopéptidos
0 horas: 10g
1 horas: 10g
3 horas: 10g
Nitrogeno
liquido
Congelación
de sedimentos
de membrana
La digestión de tripsina en solución
y la concentración de péptidos se
realizaron como se describió
anteriormente (Minkoff et al., 2014)
9. RESULTADOS
Una pantalla fosfoproteómica imparcial revela sitios de fosforilación diferencial dependientes de
ETI
Fig. 1 Schematic representation of the large-scale phosphoscreen. Individual steps
are highlighted and annotated. For further details, see the experimental
procedures in the Materials and Methods section.
Análisis cuantitativo sin etiquetas
seguido de ANOVA ( P ≤ 0.05)
10. RESULTADOS
Una pantalla fosfoproteómica imparcial revela sitios de fosforilación diferencial dependientes de ETI
(1) aquellos regulados por RPS2 activado.
10 sitios de fosforilación
(2) aquellos regulados por AvrRpt2 independientemente de la
activación de RPS2,
(3) aquellos regulados por tratamiento con Dex independientemente de
AvrRpt2 y/o RPS2,
(4) aquellos fosforilados diferencialmente únicamente dependiendo de el
genotipo de la planta.
264 sitios de fosforilación
111 sitios de fosforilación
2 sitios de fosforilación :(S280 y S283 de PIP2B)
12. Fig. 2 Heat-maps showing RESISTANT TO P. SYRINGAE-2 (RPS2)-regulated phosphorylation sites. The phosphorylation sites
shown are differentially phosphorylated on RPS2 activation (one-way ANOVA P ≤ 0.05 + t-test P ≤ 0.05). black, sites for which the
protein levels were not significantly altered amongst treatments; magenta, sites for which protein levels were significantly increased
in Dex:avrRpt2 plants on application of dexamethasone (Dex); green, sites for which protein levels were significantly decreased in
rpm1rps2/Dex:avrRpt2 plants on Dex application; orange, sites for which no peptides were detected in total protein samples; black
stars, the phosphorylation sites upregulated in both lines after Dex treatment.
15. RESULTADOS
La activación de RPS2 y FLS2 da como resultado la desfosforilación de H + -ATPasas
Fig. S2 Alignments of differentially phosphorylated sites in PIP, AHA and PIN proteins. Regions of
differentially phosphosites of AHAs(a), PIPs(b) and PINs(c) were aligned using ClustalW. The residues
in red are differentially phosphorylated residues after Dex treatment, whose total protein levels do
not change significantly in abundance (see also Table S1). The residues in orange are differentially
phosphorylated residues after dexamethasone treatment, for which total protein levels were not
detected (see also Table S1). Identified phosphopeptides are underlined within the alignment (see
also Table S6).
16. RESULTADOS
Se requiere la fosforilación de residuos comunes de RBOHD para la producción de ROS durante ETI
aclarar la importancia de estos sitios comunes de RBOHD durante PTI y ETI.
Fig. 3 Avirulent bacteria induce phosphorylation of RESPIRATORY
BURST OXIDASE HOMOLOG D (RBOHD) at specific residues.
Selected reaction
monitoring (SRM) analysis of the phosphorylation sites 6 h after
infiltration with 10mM MgCl2 solution, Pseudomonas syringae pv
tomato (Pto) DC3000
EV (empty vector), Pto DC3000 (avrRpm1) or Pto DC3000
(avrRpt2) using a triple quadrupole mass spectrometer. Values are
means SE of three
biological replicates. Different letters indicate significantly different
values at P ≤ 0.05 for S163 and S347, or at P ≤ 0.01 for S343
(one-way ANOVA, Tukey
post hoc test).
17. RESULTADOS
Se requiere la fosforilación de residuos comunes de RBOHD para la producción de ROS durante ETI
ROS
CUANDO SE INFILTRÓ CON Pto DC3000
avirulento ( avrRpm1 ) o Pto DC3000 ( avrRpt2 )
Fig. S3 RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD) phosphorylation sites S343 and S347 are required for flagellin 22
(flg22)‐inducible reactive oxygen species (ROS) burst. (a) There is no growth phenotype in rbohD and rbohD/pRBOHD:3xFLAG‐RBOHD
(WT) and rbohD pRBOHD:3xFLAG‐RBOHD (S343A/S347A) lines. (b) Phosphorylation of S343 and S347 is required for
flg22‐inducible ROS burst in Arabidopsis. Leaf discs were treated with 200 nM flg22 and the ROS production was measured over 40 min.
Values are mean ±SE (n = 8). Different letters indicate significantly different values at p≤0.001 (one way ANOVA, Tukey post hoc test). The
experiments were performed three times with similar results.
18. RESULTADOS
Fig. S4 Conservation of identified RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD) phosphorylation sites in the phosphoproteomic
screen. An alignment of Arabidopsis RBOHD (AtRBOHD), Nicotiana tabacum RBOHD (NtRBOHD), Solanum lycopersicum RBOHD (SlRBOHD)
and the rice RBOHD ortholog Oryza sative RBOHB (OsRBOHB) is provided. Phosphorylation sites whose phosphorylation were significantly
upregulated after dexamethasone treatment (one-way ANOVA+T-test p≤0.05) are shown with asterisks. Phosphorylation sites identified as
differentially phosphorylated during PTI in previous studies are indicated with a square red box.
19. RESULTADOS
Tinción de H2O2 mediada por 3,3′-diaminobencidina (DAB) en mutantes rbohD y rbohD que
expresan 3xFLAG - RBOHDWT y la variante S343A/S347A 8 h después de la infiltración con la
bacteria. Las mitades derechas de las hojas se infiltraron con solución de MgCl2 10 mM,
Pseudomonas syringae pv tomate ( Pto ) DC3000 EV, Pto DC3000 ( avrRpm1 ) o Pto DC3000 (
avrRpt2 ). Repetimos el experimento tres veces con resultados similares (se tiñeron de cuatro
a cinco hojas por genotipo cada vez).
20. RESULTADOS
Fig. 5 The bik1pbl1 double mutant does not exhibit a defect in reactive oxygen species (ROS) accumulation during effector-triggered immunity (ETI).
(a) BOTRYTIS-INDUCED KINASE-1 (BIK1) protein accumulates after infection with Pseudomonas syringae pv tomato (Pto) DC3000 (avrRpm1).
pBIK1:BIK1-HA plants were inoculated with 5mM MgCl2, Pto DC3000 empty vector (EV) or Pto DC3000 (avrRpm1) (2.5 9 107 cfu(colony-forming
units) ml1) and BIK1-HA protein amount was determined by immunoblot analyses using anti-HA antibody. Coomassie Brilliant Blue G-250 (CBB)
stain shows Rubisco protein to demonstrate equal loading. (b) H2O2 accumulation in Col-0 and bik1pbl1 mutant after inoculation with 10mM MgCl2,
Pto DC3000 EV or Pto DC3000 (avrRpm1) (2.5 9 107 cfu ml1). H2O2 accumulation was detected by 3,30- diaminobenzidine (DAB). The experiments
were performed three times
with similar results.
21. RESULTADOS
Fig. 6 RBOHD-S343A/S347A variant can complement the overactivation of immune-related gene
expression in rbohD and the semi-dwarf autoimmune phenotype of rbohDrbohF. (a) Loss of
RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) leads to increased expression of FERULOYL
COA ORTHO-HYDROXYLASE 1 (F60H1) on bacterial perception. Gene expression of F60H1 in leaves
infiltrated with Pseudomonas syringae pv tomato DC3000 (avrRpt2) (2.5 9 107 cfu (colony-forming units)
ml1) or 10mM MgCl2 solution for 24 h was measured by quantitative PCR analysis. The relative transcript
levels were calculated by normalization to the U-box housekeeping gene transcript (At5g15400). Data are
the mean SE of three technical replicates. Different letters indicate significantly different values at P ≤ 0.05
(one-way ANOVA, Tukey post hoc test). The experiments were performed three times with similar results.
(b) Immunoblot showing equal FLAG-RBOHD protein level in leaves of bohDrbohF/pRBOHD:3xFLAG-
RBOHD (wildtype, WT) and rbohDrbohF/pRBOHD:3xFLAG-RBOHD (S343A/S347A) lines. Leaf tissue of
the rbohDrbohF double mutant was used as negative control. Coomassie Brilliant Blue G-250 (CBB) shows
Rubisco protein to demonstrate equal loading. (c) Growth phenotypes of 6-wk-old plants. Bars, 1 cm.
ROS
cierre de estomas
Deposición de callosa
evita la invasión de patógenos
señalización inmune a distancia
FERULOYL COA ORTO-HIDROXILASA 1 (
F6′H1 )
escopoletina
22. RESULTADOS
Aunque las ROS están implicadas en diversas respuestas inmunitarias, no hay pruebas genéticas claras
que muestren las funciones cruciales de las ROS en la resistencia frente a patógenos avirulentos.
Se requiere la fosforilación RBOHD de S343 o S347 para la inmunidad
Fig. 7 RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) phosphorylation
sites S343 and S347 are required for effector-triggered immunity (ETI). Bacterial
growth of Pseudomonas syringae pv tomato (Pto) DC3000 (avrRpm1) (a) and Pto
DC3000 (avrRpt2) (b) in rbohD and rbohD expressing 3xFLAG-RBOHD wild-type
(WT) or the S343A/S347A variant. Bacteria were syringe infiltrated in three leaves
per plant at a concentration of 1 9105 cfu (colony-forming units) ml 1. At 3 d
postinfiltration, the leaves were harvested to determine bacterial growth. Data are
the means SD of eight replicates. Solid horizontal lines within the boxes show the
median. Different letters indicate significantly different values at P ≤ 0.01 (one-way
ANOVA, Tukey post hoc test). These experiments were repeated three times with
similar results.
23. RESULTADOS
Se requiere la fosforilación RBOHD de S343 o S347 para la inmunidad
Ensayos de enfermedad de Plectosphaerella cucumerina BMM ( PcBMM )
24. DIDCUSIONES
En apoyo de esto, encontramos que la fosforilación en residuos conservados específicos
en RBOHD es necesaria para la producción de ROS tanto en PTI como en ETI (Giacometti
et al ., 2012 ; Underwood & Somerville, 2017 ).
La inactivación de AHA1 puede conducir a la inhibición de la apertura de los estomas,
evitando así la entrada de bacterias (Yamauchi et al ., 2016 ).
Las quinasas que fosforilan los sitios de fosforilación de PTI y ETI comunes in vivo aún se
desconocen en gran medida.
También encontramos que las ROS mediadas por RBOHD contribuyen a la resistencia
contra un hongo virulento