H₂O₂ sulfenylates CHE linking local infection to establishment of systemic acquired resistance

Background

A new model to study plant-pathogen interactions

When Arabidopsis thaliana was established as a model plant for molecular biology in the 1980s, Xinnian Dong was right in the middle of the action. As postdoc, she was working with Fred Ausubel to create a new pathosystem that would allow researchers to study plant-microbe interactions with this new model organism [1]. Their team, as well as a group of colleagues working with Brian Staskawicz, eventually succeeded to establish the Arabidopsis-Pseudomonas syringae pathosystem, which has since become one of the most widely used systems to study the plant immune system [1,2].

Systemic Acquired Resistance (SAR)

For her own independent work, Dong then decided to focus on the phenomenon of systemic acquired resistance (SAR). It has long been observed that when certain parts of a plant are locally infected by a pathogen, other parts or organs, far away from the infection site, somehow acquire immunity – and not just toward the specific pathogen currently attacking the plant, but a broad resistance against a variety of different pathogens [3]. Leveraging the ease of mutant screens in Arabidopsis, Dong and her team managed to isolate a mutant, nonexpresser of PR genes 1 (npr1), that failed to induce this acquired resistance – neither in response to actual infection by Pseudomonas syringae, nor in response to known chemical inducers of SAR, salicylic acid and 2,6-dichloroisonicotinic acid [4]. Thus, this mutant provided an excellent tool to study how SAR is established in the plant, and Dong and her team set out to identify the signal that triggers SAR in tissues distal to the actual infection site.

At the time it was already clear that the phytohormone salicylic acid (SA) is one of the main inducers of SAR [5]. Upon pathogen infection, SA biosynthesis is induced at the infection site, where it activates immune pathways. This includes the drastic hypersensitive response, a form of programmed cell death in plants, which aims to stop the spread of an infection. This local, SA-induced immune response is triggered by avirulence effector proteins that the pathogen injects into the plant cells. Following this local response, the distal SAR response is launched by the plant, which is nonetheless also dependent on elevated concentrations of SA throughout the plant.

The search for the SAR-inducing signal

Thus, one early and straightforward explanation for how SAR could be induced, was that the hormone SA was itself a mobile messenger. However, when SA-silenced plant roots were grafted to wild type plant shoots, infection of the SA-deficient roots could still induce SAR and SA-accumulation in the wild type shoots, demonstrating that SA cannot be the mobile signal, but is instead locally produced in response to signal perception [6]. A second candidate for the role of the mobile signal was the reactive oxygen species (ROS) hydrogen peroxide (H₂O₂). However, no accumulation of H₂O₂ could be identified in systemic leaves following infection, and an artificially increased intracellular concentration hereof was also unable to induce SAR [7].

Thus, in 1995, Xinnian Dong pointed to this missing link between local SA-induced immunity and systemic acquired immunity in her commentary “Finding the missing pieces in the puzzle of plant disease resistance” [5]. Now, almost 30 years later, her lab reports on the identification of this missing link – and it’s no stranger [8].

In this Preprint

che – a mutant that can’t perceive the SAR-inducing signal

For their new work, Dong and her team focused on the transcription factor CCA1 HIKING EXPEDITION (CHE). CHE is a component of the circadian clock oscillator, involved in regulating diurnal stomata opening and closure, but also linking basal immunity to the circadian clock [9,10]. When the authors analyzed transcriptomic data of the che mutant following infection by Pseudomonas syringae, they noticed that the response of the mutant only differed from the wild type in systemic tissues, but not in the local, infected tissue. In the distal, systemic che tissue, SA- and pipecolic acid (Pip)-biosynthesis were no longer induced – indicating that the mutants failed to activate SAR. However, when they collected exudates from infected che tissue, and used these to treat healthy wild type tissue, the exudates still induced SAR in the healthy plant. Thus, the che mutants still appeared to produce the SAR-inducing signal, but the distal tissue was no longer able to perceive or decode it.

The CHE protein is the target of the SAR-inducing signal

CHE may therefore act in signal perception. Analyzing the CHE protein, the authors then identified a conserved cysteine residue in the DNA-binding domain of the transcription factor, that may undergo a redox reaction at a thiol moiety. And indeed, the authors could show that this cysteine can be sulfenylated when exposed to the reducing molecule H₂O₂. Further, when this residue was mutated, SA- and Pip-induction, as well as binding to the promoter of the core SA-biosynthesis gene ISOCHORISMATE SYNTHASE 1 (ICS1) were impaired, and the modified gene failed to complement the che mutant. Thus, this post-translational modification could be the activator of CHE and SAR.

H₂O₂ is the SAR-inducing signal

Importantly, sulfenylation was only observed at the specific concentration of 50 µM H₂O₂, while lower concentrations had no effect, and higher concentrations led to further oxidation and sulfinylation. This strict concentration-dependence could therefore be the explanation for why CHE only induces SAR in systemic tissue, while in local tissue, where ROS-concentrations are considerably higher, it is inactivated through further reduction and sulfinylation.

Finally, to determine the origin of the CHE-sulfenylating – and thus SAR-inducing – ROS, the authors investigated the ROS-producing NADPH-oxidase RBOHD. Following infection, ROS was produced locally and spread into the distal tissues. This was abolished in the rbohd mutant, as was CHE-sulfenylation, and binding of CHE to the ICS1 promoter as well as distal SA-biosynthesis were also impaired in the mutant.

In conclusion

It appears very likely that H₂O₂ is indeed the endogenous mobile signal that induces SAR (Figure 1): In the local, infected tissue, detection of pathogen-derived avirulence effectors induces local, effector-triggered immunity, which includes the activation of RBOHD to produce H₂O₂. At a very high local concentration, these ROS lead to the sulfinylation (not sulfenylation), and inactivation, of CHE. As a mobile signal, H₂O₂ spreads out into the distal tissue, where, at the appropriate concentration, it sulfenylates CHE thereby allowing it to bind to the ICS1 promoter, and subsequently activating SA-biosynthesis, which activates Pip-biosynthesis and triggers SAR (Figure 1).

Significance

When Arabidopsis was first established as a model to study plant-pathogen interactions, the connection between the local and systemic immune response was an early focus for researchers. One would imagine that when Xinnian Dong wrote about this connection in her 1995 article on ‘the missing pieces in the puzzle of plant disease resistance’, she did not envision that it would take 30 years to identify this link.

Intriguingly, ROS has been one of the earliest candidates to be the mobile signal, but it was ruled out, at least in part, because there were conflicting results about the concentration at which it may or may not induce SAR [7,11]. Considering that this new study shows a strict concentration-dependency for the ROS-mediated activation of CHE, these seemingly conflicting results now make sense.

Future direction and open questions

As is common for breakthrough reports, they usually open up more research questions and directions than they answer [12]. And this study is no exception.

The strict concentration-dependence of CHE-sulfenylation is one of the most interesting aspects for future research. It is conceivable that RBOHD-activation and ROS-mobility create a concentration gradient from the infection-site throughout the tissue, and that the exact position of SAR-induction is determined via this gradient (i.e., when the H₂O₂  concentration reaches 50 µM, or rather, the in planta-equivalent of the in vitro determined 50 µM). But how does SAR then spread further throughout the plant body, even to other organs? The 50 µM H₂O₂ concentration optimum, after all, is highly local. So how is SAR induced in tissue even further away, where there is even less ROS? Or is the ROS-signal further amplified, again preceding the spread of the SAR wave?

The specific post-translational modification of CHE, turning it into a SAR-inducer, is another interesting topic for future research, especially considering CHE’s second role, namely as regulator of circadian oscillations. Is it a distinct modification that turns CHE into a regulator of diurnal stomata opening? Or can CHE fulfill these functions without further modifications, whereas the reported post-translational modification diverts its activity toward SAR? In this study, the authors already show that the induction of SAR does not alter CHE binding to the promoter of the circadian clock CCA1 gene.

Finally, the role of ROS as mobile signal is most likely broader than just as an inducer of SAR. Another very interesting preprint published recently shows that ROS is also a signal that can act as messenger between two plants [13]. When two plants are infected by the same parasitic Cuscuta plant, the parasite connects its two hosts. Imaging the ROS-release in two such plants, connected via a Cuscuta bridge, Fichman et al. (2023) show that a ROS signal travels across the Cuscuta bridge from one plant to the other (it is important to note, though, that Fichman et al. (2023) can’t distinguish between the different ROS species). This may also be an attempt to activate SAR, but it seems more likely that ROS fulfills several roles as mobile messenger, which will be very interesting to untangle with future work. 

References

1. Dong X, Mindrinos M, Davis KR, Ausubel FM. Induction of Arabidopsis defense genes by virulent and avirulent Pseudomonas syringae strains and by a cloned avirulence gene. Plant Cell. 1991;3: 61–72. doi:10.1105/tpc.3.1.61
2. Whalen MC, Innes RW, Bent AF, Staskawicz BJ. Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean. Plant Cell. 1991;3: 49–59. doi:10.1105/tpc.3.1.49
3. Fu ZQ, Dong X. Systemic Acquired Resistance: Turning Local Infection into Global Defense. Annu Rev Plant Biol. 2013;64: 839–863. doi:10.1146/annurev-arplant-042811-105606
4. Cao H, Bowling SA, Gordon AS, Dong X. Characterization of an Arabidopsis Mutant That Is Nonresponsive to Inducers of Systemic Acquired Resistance. Plant Cell. 1994;6: 1583. doi:10.2307/3869945
5. Dong X. Finding the missing pieces in the puzzle of plant disease resistance. Proc Natl Acad Sci U S A. 1995;92: 7137–7139. doi:10.1073/pnas.92.16.7137
6. Vernooij B, Friedrich L, Morse A, Reist R, Kolditz-Jawhar R, Ward E, et al. Salicylic acid is not the translocated signal responsible for inducing systemic acquired resistance but is required in signal transduction. Plant Cell. 1994;6: 959–965. doi:10.1105/tpc.6.7.959
7. Neuenschwander U, Vernooij B, Friedrich L, Uknes S, Kessmann H, Ryals J. Is hydrogen peroxide a second messenger of salicylic acid in systemic acquired resistance? Plant J. 1995;8: 227–233. doi:10.1046/j.1365-313X.1995.08020227.x
8. Cao L, Yoo H, Chen T, Mwimba M, Zhang X, Dong X. H2O2 sulfenylates CHE linking local infection to establishment of systemic acquired resistance. bioRxiv. 2023; 550865. doi:10.1101/2023.07.27.550865
9. Hassidim M, Dakhiya Y, Turjeman A, Hussien D, Shor E, Anidjar A, et al. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and the Circadian Control of Stomatal Aperture. Plant Physiol. 2017;175: 1864–1877. doi:10.1104/pp.17.01214
10. Zheng X, Zhou M, Yoo H, Pruneda-Paz JL, Spivey NW, Kay SA, et al. Spatial and temporal regulation of biosynthesis of the plant immune signal salicylic acid. Proc Natl Acad Sci U S A. 2015;112: 9166–9173. doi:10.1073/pnas.1511182112
11. Chen Z, Silva H, Klessig DF. Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. Science (80- ). 1993;262: 1883–1886. doi:10.1126/science.8266079
12. Somssich IE. Closing Another Gap in the Plant SAR Puzzle. Cell. 2003;113: 815–816.
13. Fichman Y, Mudalige AK, Lee H-O, Mittler R, Park S-Y. Plant-to-plant reactive oxygen signal transmission via a Cuscuta bridge. bioRxiv. 2023; 548730. doi:10.1101/2023.07.13.548730

Tags: arabidopsis thaliana, plant biology, plant immunity, plant pathology, pseudomonas syringae, reactive oxygen species, systemic acquired resistance

doi: https://doi.org/10.1242/prelights.35451

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