Abstract

Plants are equipped with versatile pattern recognition receptors (PRRs), which monitor their external environment and elicit defensive measures upon detection of potential risk for disease. Inside the cell, receptor-like cytoplasmic kinases (RLCKs) are key components of PRR signalling, but their molecular functions and regulatory interactions are not yet fully understood. In tomato, two RLCKs, Pti1a and Pti1b, are important signalling components that relay early defence signals elicited by bacterial flagellin, a conserved pattern common to various pathogenic and non-pathogenic microbes. An important question to resolve is how plant immune reactions are regulated to prevent unnecessary defensive measures. A recent paper published in the Biochemical Journal by Giska and Martin [Biochem. J. (2019) 476, 1621–1635] reports the identification and biochemical characterization of a new tomato (Solanum lycopersicum) protein phosphatase that negatively controls early defence signalling. The phosphatase, termed pattern-triggered immunity inhibiting PP2C 1 (Pic1), negatively controls the signalling function of Pti1b and therefore holds a central position in the defence signalling network. The Pti1b–Pic1 kinase–phosphatase interaction provides mechanistic insights that forward our understanding of protein phosphatases and their importance in plant immunity.

One of the major goals in plant science is to elucidate and fortify the molecular mechanisms of plant immunity. Changing weather conditions, pathogens and herbivores can have devastating consequences in agriculture and forestry, and a significant proportion of global agricultural yield is annually lost due to plant disease [1]. To ward off biotic challenges, plants monitor their environment by plasma membrane pattern recognition receptors (PRRs), which can recognize conserved molecular patterns of microbes or host-derived molecules that arise as a consequence of pathogen-induced tissue damage [2,3]. Currently, there is increasing interest towards the application of PRRs for the generation of durable, broad-spectrum disease resistance in crops [4]. However, before biotechnological engineering of receptor signalling can become feasible, their molecular function and regulatory interactions must be thoroughly understood. Recent work by Giska and Martin [5] provides new insight into negative regulation of early defence signalling, thereby elucidating a key element in preventing excessive channelling of resources for defensive measures. To date, numerous receptor complexes, signalling cascades and transcription factors underlying plant immunity have been identified and functionally characterized [6]. However, major gaps still remain in understanding how the initial recognition of attempted infection becomes translated into appropriate defence reactions, and how the elicited responses are controlled to avoid exaggeration of defensive measures.

Inside the cell, PRRs are functionally linked with receptor-like cytoplasmic kinases (RLCKs), which mediate important roles in the relay of information to downstream signalling nodes in order to trigger appropriate defence responses against the multitude of microbial pathogens [7]. Since plant defences form a considerable sink for energy and metabolic intermediates, negative regulators that prevent unnecessary defence responses can significantly affect plant fitness and productivity. Giska and Martin [5] identified and biochemically characterized a new tomato (Solanum lycopersicum) protein phosphatase that can negatively control early defence signalling. The phosphatase, termed pattern-triggered immunity inhibiting PP2C 1 (Pic1), negatively controls the signalling function of the RLCK Pti1b, an important cytosolic protein kinase that mediates flagellin-induced signals and promotes resistance against the hemibiotrophic bacterial pathogen Pseudomonas syringae pv. tomato (Pst) [8]. These findings provide mechanistic understanding concerning the function of protein phosphatases in limiting the extent of defensive measures.

Much of the basic understanding of plant immunity has arisen from studies on the model plant Arabidopsis thaliana and its harmful biotic interactions, but there is an increasing trend towards a comprehensive understanding of immune reactions in crops. The interaction between tomato and Pst has become a widely applied model system, which has been instrumental in deciphering the molecular basis of plant disease resistance [9]. A significant research effort has led to the identification of a range of PRRs and their ligands in different plant–microbe interactions [4]. These sensory systems include receptor-like kinases and receptor-like proteins, which alarm the host cell upon detection of potential danger [3,10], eliciting an early defence response collectively referred to as pattern-triggered immunity (PTI). Arabidopsis FLAGELLIN-SENSING 2 (FLS2) is one of the best-understood PRRs and, as the name suggests, perceives a peptide flg22 that derives from bacterial flagellin [11,12]. In tomato and other Solanaceous species, the receptor kinase FLAGELLIN-SENSING 3 activates the plant immune system upon binding another flagellin-derived peptide, flgII-28 [13].

After recognizing their target, PRRs undergo dynamic structural rearrangements that allow signal initiation by the activated receptor complex. Hallmarks for PTI-associated responses include activation of NADPH oxidase-driven burst of reactive oxygen species (ROS) in the apoplast and signalling through mitogen-activated protein kinase (MAPK) and calcium-dependent protein kinase (CDPK) cascades in the cytosol [2,14,15]. The functional hierarchy and regulatory interactions between ROS-induced signals, RLCKs, MPKs and CDPKs still remain ambiguous. It is clear, however, that the downstream defence responses are orchestrated by extensive inter-pathway cross-talk that culminates at transcriptional and metabolic reprogramming, biosynthesis of deterring secondary metabolites and reinforcement of the host cell wall [2]. Besides the first layer of defence activation through PTI, plants commonly undergo a second, more durable wave of defensive measures termed effector-triggered immunity. Pathogens secrete effectors to manipulate the host cells in order to facilitate pathogenesis. As a counter measure, plants can recognize the presence of pathogen effectors and mount stronger defensive measures that are commonly accompanied by the hypersensitive response [2].

Reflecting the centrality of protein phosphorylation as a means for triggering defensive measures, controlled protein dephosphorylation by protein phosphatases provides an important regulatory mechanism that can control the perception, relay and duration of external signals. An increasing number of different types of protein phosphatases that negatively regulate immune responses has been identified and functionally characterized. For example, Segonzac et al. [16] showed that a heterotrimeric protein phosphatase 2A (PP2A) complex negatively regulates the activity of BRI1-ASSOCIATED KINASE 1 (BAK1), an essential co-receptor in PRR signalling. The cytosolic PRR target BOTRYTIS-INDUCED KINASE (BIK1), in turn, is negatively controlled by a monomeric protein phosphatase PP2C38 [17]. Within intracellular regulatory networks, PP2C-type MAP KINASE PHOSPHATASEs are well-known for their roles in controlling immunity signalling through MPK3 and MPK6 [18,19], while PP2A targets metabolic enzymes to mitigate chemical defences in the absence of infection [20].

Giska and Martin [5] add to the understanding of reversible phosphorylation by identifying the tomato RLCK Pti1b as a regulatory target for the PP2C-type protein phosphatase Pic1. The initial discovery was made by in vivo co-immunoprecipitation assays coupled with mass spectrometry, which revealed that Pti1b co-purified with Pic1 from Nicotiana benthamiana leaves [5]. Further in vitro characterization with a series of Pti1b variants and Pic1 produced in Escherichia coli confirmed the protein interaction, which was demonstrated to be independent of the phosphorylation status of Pti1b [5]. Subsequent assays with Pti1b, together with active and inactive variants of Pic1 showed that Pic1 dephosphorylates Pti1b both in vitro and in vivo [5].

Pti1 seems to act early in PTI signalling by inducing ROS production in response to flagellin perception, thereby influencing the expression of defence-related genes and enhancing resistance to Pst [5]. Pti1b localizes to the cell periphery, but whether it physically associates with FLS2 or FLS3, or operates downstream of these complexes, remains to be established. Whatever the case, by dephosphorylating the Pti1b kinase, Pic1 can essentially limit MAMP-induced ROS burst and the activation state of MAMP-triggered signalling [5]. This conclusion is also supported by the finding that the tomato gene encoding Pic1 becomes induced in response to treatment with MAMPs [5].

Plant immunity is governed by converging signalling networks where protein kinases and phosphatases collectively shape the optimal outcome of defensive measures. It is therefore of interest that even though silencing of Pti1 kinases weakens flagellin-induced ROS production, it has no significant effect on the downstream MAPK activation. Future work on Pti1 signalling may shed light on the concurrent initiation of multiple downstream signalling events linked to PRRs, which is critical in establishing appropriate cellular responses [27]. Likewise, accurate control of ROS production and defence signalling is critical in preventing unnecessary cell death or investment of resources to energy-consuming metabolic changes, such as biosynthesis of secondary metabolites. Connecting protein kinases to their counteracting protein phosphatases, revealing their target proteins, and understanding the physiological importance of the regulatory interactions, therefore, represent outstanding future research questions in plant biology.

The biochemical work on Pic1 provides novel insights into plant signalling networks [5]. While protein kinases as positive mediators of plant immune reactions have been well studied, protein phosphatases still remain less well understood. It seems clear that in the Pti1b–Pic1 interaction the phosphatase regulates the kinase, but an interesting question that warrants future examination is whether the phosphatase Pic1 is also a target for regulation by reversible phosphorylation. In addition, the work opens new research lines for identification and analysis of other Pic1-regulated pathways and their roles in plant physiology.

Abbreviations

     
  • BAK1

    BRI1-ASSOCIATED KINASE 1

  •  
  • BIK1

    BOTRYTIS-INDUCED KINASE 1

  •  
  • CDPK

    calcium-dependent protein kinase

  •  
  • FLS

    FLAGELLIN-SENSING

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • PP2A

    protein phosphatase 2A

  •  
  • PP2C

    protein phosphatase 2C

  •  
  • PRR

    pattern recognition receptor

  •  
  • Pst

    Pseudomonas syringae pv. tomato

  •  
  • PTI

    pattern-triggered immunity

  •  
  • RLCK

    receptor-like cytoplasmic kinase

  •  
  • ROS

    reactive oxygen species

Author Contribution

The manuscript was written by the author.

Acknowledgements

The author acknowledges the Academy of Finland (307719 and 307335) for financial support.

Competing Interests

The Author declares that there are no competing interests associated with this manuscript.

References

References
1
Strange
,
R.N.
and
Scott
,
P.R.
(
2005
)
Plant disease: a threat to global food security
.
Annu. Rev. Phytopathol.
43
,
83
116
2
Boller
,
T.
and
Felix
,
G.
(
2009
)
A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors
.
Annu. Rev. Plant Biol.
60
,
379
406
3
Macho
,
A.P.
and
Zipfel
,
C.
(
2014
)
Plant PRRs and the activation of innate immune signaling
.
Mol. Cell
54
,
263
272
4
Boutrot
,
F.
and
Zipfel
,
C.
(
2017
)
Function, discovery, and exploitation of plant pattern recognition receptors for broad-spectrum disease resistance
.
Annu. Rev. Phytopathol.
55
,
257
286
5
Giska
,
F.
and
Martin
,
G.B.
(
2019
)
PP2C phosphatase Pic1 negatively regulates phosphorylation status of Pti1b kinase, a regulator of flagellin-triggered immunity in tomato
.
Biochem. J.
476
,
1621
1635
6
van der Burgh
,
A.M.
and
Joosten
,
M.H.A.J.
(
2019
)
Plant immunity: thinking outside and inside the box
.
Trends Plant Sci.
24
,
587
601
7
Liang
,
X.
and
Zhou
,
J.M.
(
2018
)
Receptor-like cytoplasmic kinases: central players in plant receptor kinase-mediated signaling
.
Annu. Rev. Plant Biol.
69
,
267
299
8
Schwizer
,
S.
,
Kraus
,
C.M.
,
Dunham
,
D.M.
,
Zheng
,
Y.
,
Fernandez-Pozo
,
N.
,
Pombo
,
M.A.
et al.  (
2017
)
The tomato kinase Pti1 contributes to production of reactive oxygen species in response to two flagellin-derived peptides and promotes resistance to Pseudomonas syringae infection
.
Mol. Plant Microbe Interact.
30
,
725
738
9
Pedley
,
K.F.
and
Martin
,
G.B.
(
2003
)
Molecular basis of Pto-mediated resistance to bacterial speck disease in tomato
.
Annu. Rev. Phytopathol.
41
,
215
243
10
Gust
,
A.A.
and
Felix
,
G.
(
2014
)
Receptor like proteins associate with SOBIR1-type of adaptors to form bimolecular receptor kinases
.
Curr. Opin. Plant Biol.
21
,
104
111
11
Gomez-Gomez
,
L.
and
Boller
,
T.
(
2000
)
FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis
.
Mol Cell.
5
,
1003
1011
12
Zipfel
,
C.
,
Robatzek
,
S.
,
Navarro
,
L.
,
Oakeley
,
E.J.
,
Jones
,
J.D.
,
Felix
,
G.
et al.  (
2004
)
Bacterial disease resistance in Arabidopsis through flagellin perception
.
Nature
428
,
764
767
13
Hind
,
S.R.
,
Strickler
,
S.R.
,
Boyle
,
P.C.
,
Dunham
,
D.M.
,
Bao
,
Z.
,
O'Doherty
,
I.M.
et al.  (
2016
)
Tomato receptor FLAGELLIN-SENSING 3 binds flgII-28 and activates the plant immune system
.
Nat. Plants
2
,
16128
14
Asai
,
T.
,
Tena
,
G.
,
Plotnikova
,
J.
,
Willmann
,
M.R.
,
Chiu
,
W.-L.
,
Gomez-Gomez
,
L.
et al.  (
2002
)
MAP kinase signalling cascade in Arabidopsis innate immunity
.
Nature
415
,
977
983
15
Boudsocq
,
M.
,
Willmann
,
M.R.
,
McCormack
,
M.
,
Lee
,
H.
,
Shan
,
L.
,
He
,
P.
et al.  (
2010
)
Differential innate immune signalling via Ca2+ sensor protein kinases
.
Nature
464
,
418
422
16
Segonzac
,
C.
,
Macho
,
A.P.
,
Sanmartín
,
M.
,
Ntoukakis
,
V.
,
Sánchez-Serrano
,
J.J.
and
Zipfel
,
C.
(
2014
)
Negative control of BAK1 by protein phosphatase 2A during plant innate immunity
.
EMBO J.
33
,
1
11
17
Couto
,
D.
,
Niebergall
,
R.
,
Liang
,
X.
,
Bucherl
,
C.A.
,
Sklenar
,
J.
,
Macho
,
A.P.
et al.  (
2016
)
The Arabidopsis protein phosphatase PP2C38 negatively regulates the central immune kinase BIK1
.
PLoS Pathog.
12
,
e1005811
18
Lumbreras
,
V.
,
Vilela
,
B.
,
Irar
,
S.
,
Solé
,
M.
,
Capellades
,
M.
,
Valls
,
M.
et al.  (
2010
)
MAPK phosphatase MKP2 mediates disease responses in Arabidopsis and functionally interacts with MPK3 and MPK6
.
Plant J.
63
,
1017
1030
19
Bartels
,
S.
,
Anderson
,
J.C.
,
Gonzalez Besteiro
,
M.A.
,
Carreri
,
A.
,
Hirt
,
H.
,
Buchala
,
A.
et al.  (
2009
)
MAP kinase phosphatase1 and protein tyrosine phosphatase1 are repressors of salicylic acid synthesis and SNC1-mediated responses in Arabidopsis
.
Plant Cell
21
,
2884
2897
20
Durian
,
G.
,
Rahikainen
,
M.
,
Alegre Garcia
,
S.
,
Brosché
,
M.
and
Kangasjärvi
,
S.
(
2016
)
Protein phosphatase 2A in the regulatory network underlying biotic stress resistance in plants
.
Front. Plant Sci.
7
,
812