Introduction
The plant immune system is multi-layered and complex. It traditionally
comprises three modules; microbe associated molecular pattern
(MAMP)-triggered immunity (MTI), effector-triggered immunity (ETI) and
systemic acquired resistance (SAR) (Jones and Dangl, 2006; Shineet al. , 2019). The initial layer of defence, MTI, provides
broad-spectrum defence against a diverse range of pathogens and has
recently been shown to be involved in potentiating ETI responses, which
can in turn reinforce MTI (Lu and Tsuda, 2021; Ngou et al. , 2021;
Nguyen et al. , 2021; Yuan et al. , 2021). Classical
pathogen cell surface receptors comprise transmembrane receptor-like
kinases (RLKs) or receptor-like proteins (RLPs) including FLAGELLIN
SENSING 2 (FLS2), EF-Tu RECEPTOR (EFR) and CHITIN ELICITOR RECEPTOR
KINASE 1 (CERK1-2) which, respectively, detect flagellin and elongation
factor thermo-unstable (EF-Tu) from bacterial pathogens and chitin from
fungi (Yu et al. , 2017). However, an increasing number of MAMPs
associated with a diverse range of pathogens have been identified
(Noman, Aqeel and Lou, 2019). In addition, cell surface receptors can
detect plant derived damage associated molecular patterns (DAMPs) found
within extracellular spaces. Amongst DAMP receptors are the
well-characterised RLKs, PEP RECEPTOR 1 (PEPR1) and PEPR2 which detect
plant elicitor peptides, Peps. PEPR1, recognises Peps1-6 while PEPR2
recognises only Pep1 and Pep2 (Yamaguchi, Pearce and Ryan, 2006;
Yamaguchi et al. , 2010). These Peps are cleaved from the
C-terminus of plant PROPEPs during cell damage and the transcripts of
PROPEP1-3 are induced by defence-related hormones methyl salicylate
(MeSA) and methyl jasmonate (MeJA) (Huffaker, Pearce and Ryan, 2006;
Yamaguchi et al. , 2010).
The pattern recognition receptors (PRRs), FLS2, EFR and PEPR1/2, are
cell membrane localised and contain extracellular leucine rich repeat
(LRR) surfaces where their ligands bind. Upon peptide detection by PRRs,
co-receptors are recruited and bind to PRRs (and in some cases the
ligand). The well characterised co-receptor Brassinosteroid Insensitive
1 (BRI1)-associated receptor kinase 1 (BAK1) belongs to the somatic
embryogenesis receptor-like kinase family (SERK) which contains five
members, one of which, SERK4/BKK1 (BAK1-LIKE 1), has high sequence
similarity to BAK1 and has functional redundancy (He et al. ,
2007). While BAK1 was first identified as a co-receptor for the
Brassinosteroid receptor BRI1, involved in cell growth and division, it
has become widely known for its role in plant immunity as plants
containing the reduced function bak1-5 allele have impaired FLS2,
EFR and PEPR receptor function (Roux et al. , 2011; Schwessingeret al. , 2011). In contrast, bkk1-1 still exhibits a
reactive oxygen species (ROS) burst and MAP Kinase (MPK3, MPK4 and MPK6)
activation, that is comparable to wild type plants, when treated with
flg22 or elf18. However, the bak1-5/bkk1-1 plants show minimal
ROS and no MAPK (mitogen-activated protein kinase) activation in
response to these PAMPs (Zipfel et al. , 2006; Roux et al. ,
2011).
MTI triggers rapid calcium signalling, ROS and MAPK signalling cascades
all of which involve plasma membrane to nuclear signalling (Noman, Aqeel
and Lou, 2019). Microbes successful in colonisation secrete effectors to
inter- or intracellular locations, which can dampen MTI signalling.
Examples of such effector triggered suppression (ETS) include the AvrPto
effector from Pseudomonas syringae which interacts with the PRRs
FLS2 and EFR to dampen MTI in Arabidopsis thaliana (Xianget al. , 2008) and AvrE from P. syringae and the maize
pathogen Pantoea stewartii subsp. Stewartia which targets
protein phosphatase 2A (PP2A) complexes in order to dampen MTI (Jinet al. , 2016).
Effectors collectively target an array of plant immune signalling
components, many of which still remain elusive. Some effectors are
directly or indirectly recognised by cytoplasmic receptors, most often
belonging to the nucleotide-binding leucine-rich repeat receptors (NLRs)
class, activating a second immune response, ETI (Jones and Dangl, 2006).
There are three major classes of NLRs, the first two classically defined
by their N-terminal; Toll-like, Interleukin-1 receptor domain TIR-NLRs
(TNLs), coiled-coil domain CC-NLRs (CNLs). More recently the Resistance
to Powdery Mildew 8 (RPW8) CC-NLR class (RNLs) (Jones, Vance and Dangl,
2016; Zhong and Cheng, 2016) have been described which act as “helper”
NLRs for TNL and CNL “sensor” NLRs (Lu and Tsuda, 2021; Nguyenet al. , 2021; Maruta et al. , 2022). Interaction of an
effector and NLR is usually associated with the macroscopic development
of the hypersensitive response which restricts pathogen growth.
Classically, MTI research has centred around signal transduction
pathways originating from the plasma membrane and activating nuclear
transcription however, it is becoming increasingly recognised that
chloroplasts are a key hub of immune signalling (Kachroo, Burch-Smith
and Grant, 2021; Littlejohn et al. , 2021). Chloroplasts play a
central role in integrating environmental signals and maintaining
cellular homeostasis via retrograde signalling (de Souza, Wang and
Dehesh, 2017; Breeze and Mullineaux, 2022). Relevant to host immune
signalling, chloroplasts are also the site of chloroplastic ROS (cROS)
generation and synthesis of defence hormone precursors, jasmonic acid
(JA), salicylic acid (SA) and abscisic acid (ABA) (Littlejohn et
al. , 2021). A key early MTI response is the rapid ROS generation, an
apoplastic localised respiratory burst, primarily generated by RBOHD, a
member of the nicotinamide adenine dinucleotide phosphate (NADPH)
oxidase homologue (RBOH) family (Miller et al. , 2009). Activating
MTI using an effector secretion deficient strain of P. syringaepv. tomato strain DC3000 (DC3000hrpA ) also rapidly
generates cROS production in A. thaliana , which is attenuated in
the virulent DC3000 strain, shortly after effector delivery (de Torres
Zabala et al. , 2015).
Concomitant with differences in cROS production during infection between
the P. syringae strains DC3000 and DC3000hrpA , global
transcriptome profiling of A. thaliana revealed significant
alterations of nuclear encoded chloroplast genes (NECG s).
Remarkably, NECGs, represent ~10% of all
differentially upregulated genes and ~30% of those
significantly down regulated (de Torres Zabala et al. , 2015)
during early MTI responses despite NECGs collectively account for
only ~14% of the transcriptome. Superimposed on this,
effector delivery (2-3 hour post infection; hpi) caused transcriptional
reprogramming of NECGs , suggesting ETS also targets NECGexpression (de Torres Zabala et al. , 2015). These molecular
signatures are reflected by physiological changes between DC3000 and
DC3000hrpA challenge as evidenced by quantifying net
photosynthetic CO2 assimilation (Asat)
and chlorophyll fluorescence imaging parameters associated with electron
transport during photosynthesis. DC3000 but not DC3000hrpAchallenge induced a decrease in CO2 assimilation,
maximum dark-adapted quantum efficiency
(Fv/Fm ), maximum operating
efficiency of photosystem II (PSII)
(Fv′/Fm′ ) and the efficiency with
which light absorbed by PSII is used for quinone acceptor (QA) reduction
and linear electron transport
(Fq′/Fm’ ) (de Torres Zabalaet al. , 2015). In addition, DC3000 infection elicited an increase
in Non-Photochemical Quenching (NPQ) and PSII redox state (qL;
(Fq′/Fv′)/(Fo′/F′ ))
compared to DC3000hrpA (de Torres Zabala et al. , 2015). qL
estimates the percentage of open PSII centres and the oxidation state of
the primary PSII QA (Baker, 2008). An increase in qL suggests a decrease
in electron transport from PSII. Thus, virulent pathogens can radically
alter chloroplast physiological functions as part of their virulence
strategy.
De novo induction of the plant hormone ABA during DC3000
infection contributes to ETS (de Torres-Zabala et al. , 2007) and
was also recently shown to play a significant role in modulating
chloroplast function. DC3000 induced suppression ofFv/Fm was accelerated by
co-infiltration of 10 µM ABA, effectively phenocopying DC3000 challenge
of the Arabidopsis ABA hypersensitive protein phosphatase 2C
(PP2C) abi1/abi2/hab1 triple mutant. By contrast, the ABA
deficient Arabidopsis aldehyde oxidase 3 (aao3 )
mutant restricted DC3000 suppression ofFv/Fm (de Torres Zabala et
al. , 2015). Collectively these data show that the chloroplast is
targeted early in pathogen infection and prior to bacterial
multiplication, one of the earliest events being suppression of cROS.
This study focussed on how well characterised PRRs and co-receptors
modulated chloroplast physiology, including accessing whether diverse
signalling pathways converged to similarly modulate chloroplast
function. Here we comprehensively examine chlorophyll fluorescence
dynamics and the impact on attenuating chloroplast cROS. We show that
pre-treatment of receptor mutant plants with MAMP and DAMP peptides
generally offer protection against effector modulation of chlorophyll
fluorescence but surprisingly, fls2 plants pre-treated with
chitin fail to provide such protection. The double mutant of the MTI
co-receptors bak1-5/bkk1-1 exhibits a remarkable decrease inFv/Fm compared to control plants
during infection, underlining the importance of MTI mediated signalling
in underpinning chloroplast immunity. Expanding these findings to better
understand the role of ABA and abiotic stress in chloroplast immunity we
found that high light overrides the protection offered by MAMPs on
wild-type plants.
Materials and Methods
Arabidopsis growth conditions. Arabidopsis thalianaseeds were sown in a compost mix comprising Levingston F2 compost + sand
(LEV206):vermiculite (medium grade) mixed in a 6:1 ratio. Plants were
grown in a controlled environment growth chamber under a 10 h day (21
°C; 120 µmol m-2s-1) and 14 h night
(21 °C) with relative humidity of 65% for 5–6 weeks prior to use.
Arabidopsis peptide treatment. Pre-treatment of plants was
conducted 16 h prior to bacterial challenge. Co-infiltration experiments
were conducted by mixing the peptide or hormone of interest with the
bacterial culture to attain the required final concentration and
OD600 prior to infiltration. Concentrations of peptides
or hormones were as follows; 1 µM of flg22, elf18, Pep1, Pep2 and Pep3,
100 µg/ml of Chitin (Sigma - C9752) and 10 or 100 µM ABA.
H2O was used as mock for pre-treatments.
Bacterial growth, maintenance and inoculation.Pseudomonas syringaestrains were grown on solid Kings B media
containing appropriate
antibiotics as described (Truman, de Zabala and Grant, 2006). For
inoculation, overnight cultures were grown with
shaking (200 rpm) at 28 °C. Cells
were harvested (2,500 g × 7 min), washed and re-suspended in 10 mM
MgCl2. Cell density was adjusted to
OD600 0.15 (∼0.75 × 108 colony forming
units (cfu) ml-1) for fluorescence imaging and confocal
microscopy or
OD600 0.0002 for
growth assays. All growth assays and ROS imaging experiments were
performed at least three times. All fluorescence imaging experiments were
performed at least four times.
Chlorophyll fluorescence imaging. Photosystem II chlorophyll
fluorescence imaging of Arabidopsis rosettes was performed with a CF
Imager (Technologica Ltd, Colchester, UK). Normal light cycle;plants were placed in the chamber for 40 min post-inoculation and then
dark adapted for 20 min. This was followed by a saturating light pulse
(6,349 µmol m-2s-1 for 0.8 s) to
obtain maximum dark-adapted fluorescence (Fm ).
Actinic light (120 µmol m-2s-1 –
the same as plant growth light intensity) was then applied for 15 min,
followed by a saturating pulse to obtain maximum light adapted
fluorescence (Fm′ ). The plants remained in actinic
light for a further 24 min, then returning to a dark period of 20 min.
This cycle (59 min duration) was repeated 23 times. High light
cycle; plants were placed in the chamber for 40 min post-inoculation
and then dark adapted for 20 min. This was followed by a saturating
light pulse (6,349 µmol m-2s-1 for
0.8 s) to obtain maximum dark-adapted fluorescence
(Fm ). High light (650 µmol
m-2s-1) was then applied for 15 min,
followed by 3 saturating light pulses 5 minutes apart to obtain maximum
light adapted fluorescence (Fm′ ). The plants
remained in high light for a further 150 min then returned to a 20 min
dark phase. This cycle (200 min duration) was repeated 8 times.Fm , Fm′ andFo (minimal fluorescence with fully oxidized PSII
centres) were used to calculate chlorophyll fluorescence parameters
related to photosystem II: Fv/Fm(maximum dark-adapted quantum efficiency) and non-photochemical quenching
(NPQ). These values were calculated as described by (Baker, 2008).
Bacterial growth measurements. Three leaves per plants (6
plants total) were syringe infiltrated with bacteria,
OD600 0.0002, and placed either under high light (450
µmol m-2s-1 or 600 µmol
m-2s-1) or normal light (120 µmol
m-2s-1) for 4 days. Three
independent leaf discs per plant were excised and homogenised using a
Tissue Lyser (Qiagen). Serial dilutions were spotted on
Kings B media and colonies were
counted 24 hpi.
Confocal microscopy. Col-0 plants were pre-treated with either
water or peptide 16 h prior to bacterial challenge, then 3.5 hpi leaves
were detached and floated, adaxial surface upwards, in a solution of 10
mM MgCl2 containing 10 μM 2′7′-dichlorodihydrofluorescein
diacetate (H2DCF-DA; Enzo) for 40 min, then washed for
20 min in 10 mM MgCl2 before imaging. Samples were
mounted in perfluorodecalin (Littlejohn et al. , 2010) and images
were captured on a Zeiss 880 using a 40× oil immersion lens. Argon laser
excitation at 488 nm and an emission window of 512–527 nm was used to
capture the dichlorofluorescein
(DCF) signal. Chloroplast fluorescence was measured at 659–679 nm.