Discussion
We had previously shown that MTI significantly alters the expression of photosynthetic and nuclear encoded chloroplast localised genes (NECGs ) within the first 2 h of challenge with the T3SS deficient non-pathogenic DC3000hrpA (de Torres Zabala et al. , 2015; Lewis et al. , 2015). Notably, this MTI response results in a strong suppression of NECGs yet does not significantly reduce maximum dark-adapted quantum efficiency (Fv/Fm ) of PSII (de Torres Zabalaet al. , 2015) compared to mock challenge. In comparison, virulent DC3000 can deliver effectors within 2-3 hpi, and strongly suppressesFv/Fm as well as attenuating MTI induced cROS (de Torres Zabala et al. , 2015). Notably, DC3000 significantly reconfigures the expression of NECG within 3-4 h of infection (de Torres Zabala et al. , 2015), the timing of which coincides with the delivery of effectors into the plant cell.
Priming of plants to reduce bacterial colonisation has been previously demonstrated. Zipfel et al showed that A. thaliana Col-0 plants primed with flg22 or elf18 have reduced bacterial growth after infection with DC3000 compared to mock primed plants (Zipfel et al. , 2006). In addition, Wan et al showed that chitin pre-treatment also protectsA. thaliana against DC3000 multiplication (Wan et al. , 2008). Thus, PRRs signal via a common pathway to induce MTI responses such as callose deposition, ROS and MAP Kinase activation. Activated MTI functions across pathogen classes, e.g. the fungal MAMP chitin can prime a plant against bacterial infection (Nühse et al. , 2000). Here we investigated the impact of such priming on the chloroplast as photosynthetic genes are significantly altered during disease and early immune signalling (Kachroo, Burch-Smith and Grant, 2021; Littlejohnet al. , 2021). Our data show that priming with flg22, elf18 or chitin fully attenuates suppression of the maximum dark-adapted quantum efficiency of PSII (Fv/Fm ) by DC3000. Experiments with the broad-spectrum ROS and NOS (reactive nitrogen species) stain H2DCF-DA show that this protection extends in part to restricting DC3000 suppression of cROS within primed leaves (Figure 3) (de Torres Zabala et al. , 2015). In general, plants that have lost a MAMP; FLS2, EFR, CERK1-2 or DAMP receptor, PepR1-1x2-1 can sustain normalFv/Fm during bacterial infection by priming with an alternative M/DAMP i.e., efr , cerk1-2and pepR1-1x2-1 plants retain normalFv/Fm with flg22 pre-treatment (Figures 1, 2, 5E, F and 9). We did observe however that Pep1 and 3 provided reduced attenuation ofFv/Fm suppression in fls2plants compared to Col-0 (Sup Fig 2) and unexpectedly, chitin failed to protect Fv/Fm suppression infls2 plants (Figure 2E, F and 9) whereas efr plants are protected by chitin treatment (Figure 2C, D and 9). These findings highlight specificity between initial downstream signalling through different PRRs. These data suggest that the activation of immune signals transduced by PepR1/PepR2 and CERK1 are possibly not sufficiently strong to protect against bacterial infection in the absence of FLS2. Notably, in efr mutants pre-treated with chitin, FLS2 activation could over-ride those chloroplast processes targeted by bacteria during infection. These data suggest that there may be a requirement for pre-formed complexes with co-receptors to attenuate chloroplast immune priming.
PRRs represent the first line of induced defence and most require homo or heterodimerisation with a receptor for effective immune signalling. Chitin induces the dimerization and cross-linking of AtCERK1 which is required for immune signalling (Liu et al. , 2012). By comparison, FLS2 and EFR are known to interact with co-receptors BAK1 or BKK1, members of the SERK (SOMATIC EMBRYOGENESIS RECEPTOR KINASEs) protein family. Perception of flg22 or elf18 by their ligand leads to phosphorylation of their intracellular kinase domains and induction of downstream immune signals (Zhang and Zhou, 2010). BAK1 was originally identified as the co-receptor for the Brassinosteroid (BR) cell surface receptor BRI1. MTI is impaired in bak1-5 in response to flg22, elf18 or Pep1, leading to a reduced ROS burst and dampened MAPK activation (Roux et al. , 2011). However, this mutant is not impaired in BR signalling (Schwessinger et al. , 2011). In contrast, the ROS burst and MAPK responses to flg22, elf18 or Pep1 elicitation in a loss of function bkk1-1 mutant are similar to wild type (Roux et al. , 2011). Priming of either bkk1-1 orbak1-5 individual mutants with flg22 and elf18 shows no suppression of Fv/Fm indicating that these peptides can protect the PSII function from bacterial infection (Figure 4 and 9). This is comparable to the immune response functions observed for bkk1-1 and the BR responses observed forbak1-5 (Roux et al. , 2011; Schwessinger et al. , 2011). Chitin peptide priming preventedFv/Fm suppression by DC3000 inbkk1-1 plants but provided only partial protection inbak1-5 plants (Sup Fig 1a-d, and Fig 9), consistent with the compromised immune signalling in bak1-5 . The double mutantbkk1-1/bak1-5 has dramatically reduced immune responses to flg22 and elf18 elicitation (Roux et al. , 2011). Here we demonstrated that at the level of chloroplast function, priming with flg22, elf18 or chitin offered no protection. Rather, we measure a quantitative hyper reduction in Fv/Fm in comparison to Col-0 plants (Figure 4 and 9, Sup Fig 1d, e). The fact that chitin only provided partial protection to bak1-5 and no protection tobkk1-1/bak1-5 plants is of interest since, to date, the LysM containing chitin receptor CERK1 is not known to use BAK1 or BKK1 for signalling (Liu et al. , 2012; Yasuda, Okada and Saijo, 2017). These data suggest that additional downstream signals linked to BAK1 are required for CERK1 signalling.
Both the chloroplast and light have an impact on plant resistance. Exposure of plants to high light causes rapid changes in nuclear gene expression in a photosynthesis-dependent manner and is associated with chloroplast-to-nucleus retrograde signalling (Suzuki et al. , 2012; Vogel et al. , 2014; Exposito-Rodriguez et al. , 2017). A 1 h high light treatment of Nicotiana benthamianareduced Fv/Fm from 0.7 to 0.5 (Exposito-Rodriguez et al. , 2017), a more significant drop than we see with A. thaliana over a 3.5 h period (Figure 6C), most likely consistent with the higher light intensity of 1000 µmol m-2s-1 compared to 650 µmol m-2s-1 used in this study. Notably, this drop in Fv/Fm was accompanied by a 50% increase in H2O2(Exposito-Rodriguez et al. , 2017). A genetically encoded H2O2 reporter localised to the stroma and nucleus revealed that high light treatment induced H2O2 production in these organelles for up to 1 h, and critically, the increase in nuclear H2O2 was dependant on electrons from the chloroplast (Exposito-Rodriguez et al. , 2017). High light conditions also induced perinuclear clustering of 7-8 chloroplast per nucleus, a similar observation as has been reported for plant-virus interactions (Caplan et al. , 2015; Ding et al. , 2019). It is predicted that this physical localisation facilitates the rapid diffusion of H2O2 from chloroplast to nucleus which elicits an alteration in nuclear gene expression (Exposito-Rodriguez et al. , 2017). Here we examined the effect of high light on P. syringae infection with the addition of peptide pre-treatments and ABA signalling. Our results show that high light has a synergistic effect with effector mediated suppression ofFv/Fm . Critically, MAMP pre-treatment or ABA co-infiltration fail to attenuate this suppression during DC3000 infection (Figure 6A-F, 7F, G and 9).Fv/Fm levels during a DC3000hrpA infection also reduced significantly during the first 6 h of high light but recovered to 0.7, compared to 0.75 under normal light (Figure 6A-D). Furthermore, the co-receptor double mutant,bkk1-1/bak1-5 , also showed increasedFv/Fm suppression compared to wild type following DC3000 challenge which was accentuated under high light (Figure 6 G-I). Strikingly, contrary to expectations given the elevated H2O2 production, Col-0,aao3 and bkk1-1/bak1-5 lines all showed a significant increase in bacterial growth under high light whereas the hypersensitiveabi1/abi2/hab1 mutant and flg22 insensitive mutant fls2showed no increase in susceptibility to DC3000 infection compared to Col-0 under high light (Figure 6I, 7C). How and why these lines are insensitive to high light conditions warrants further investigation.
Complex plant hormone synthesis and signalling crosstalk play an important role in the outcome of plant disease and defence responses. Both salicylic acid (SA) and jasmonic acid (JA) are considered key hormones involved in plant immunity however, it has become apparent in recent years that ABA has a significant role to play in hormone manipulation during pathogen infection (Robert-Seilaniantz, Grant and Jones, 2011). Many organisms produce ABA, from cyanobacteria and fungi to humans, with kingdom specific synthesis pathways. In plants ABA is synthesised from carotenoids within the chloroplast, with the final two enzymatic reactions in the cytosol (Schwartz, Qin and Zeevaart, 2003; Finkelstein, 2013). As part of its virulence strategy, P. syringae induces de novo ABA biosynthesis in planta and this acts in part by suppressing SA biosynthesis and SA-mediated defences to aid disease progression (de Torres-Zabala et al. , 2007; De Torres Zabala et al. , 2009; Salomon et al. , 2014). Application of exogenous ABA (or coronatine) also induces the expression of the genes encoding three protein phosphatases 2C (PP2Cs), HAI1, HAI2, and HAI3 all of which interact with and inactivate MPK3 and MPK6, resulting in ABA-mediated MPK3/MPK6 immune suppression (Mineet al. , 2017). The PP2C triple mutant, abi1/abi2/hab1 is ABA hypersensitive and has enhanced susceptibility to DC3000 whereas the ABA biosynthetic mutant aao3 shows enhanced disease resistance (de Torres-Zabala et al. , 2007; De Torres Zabala et al. , 2009). Chlorophyll fluorescence allows dissection of the dynamics of these mutants during DC3000 infection, with the triple mutant exhibiting a stronger suppression of Fv/Fm (and a faster increase in NPQ) while the converse is true for theaao3 mutant compared to Col-0 (Figure 7A, B, Supp Figure 3) (de Torres Zabala et al. , 2015). Notably, endogenous and exogenous ABA differentially impact apoplastic ROS production, with flg22 challenge of transgenic lines overexpressing ABA resulting in increased apoplastic H2O2 production, whereas plants with reduced ABA levels produced less apoplastic H2O2 following flg22 treatment (Tanet al. , 2019). By contrast, ABA pre-treatment resulted in a reduction in flg22 induced apoplastic H2O2 indicating that endogenous and exogenous ABA function differently during MAMP-induced apoplastic ROS burst in A. thaliana (Tan et al. , 2019). During a DC3000hrpA infection, cROS is produced 3-4hpi, whereas DC3000 infection suppresses cROS, but not when primed with elf18 or flg22 (Figure 3A, B) (de Torres Zabala et al. , 2015). Unexpectedly, cROS generation appeared ABA dose dependent, as leaves co-infiltrated with DC3000 and 10 µM ABA elicited a faster decrease ofFv/Fm (similar to the hypersensitive abi1/abi2/hab1 mutants).
Collectively, these data show thatFv/Fm is a reliable, quantitative, real-time indication of pathogen infection and that abiotic factors affecting chloroplast functions e.g., high light and ABA (induced during drought and other abiotic stresses) generally result in reduced tolerance to bacterial infection.