NITROGEN LIMITATION ADAPTATION functions as a negative regulator of Arabidopsis immunity

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   PREreview

   May 3, 2022

   This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6513650.

   Phosphorus is an essential macro-nutrient and its availability to plants is known to modulate their immunity via the Phosphate Starvation Response (PSR). This was shown in Castrillo et al. 20171, where the authors demonstrated that an Arabidopsis thaliana (hereafter Arabidopsis) phr1 phl1 double mutant unable to deploy the PSR showed, under inorganic phosphate (Pi) limiting conditions, enhanced immunity levels transcriptionally and enhanced resistance to two biotrophic leaf pathogens. Genes highlighted in the transcriptome analysis notably include several transcription factors responsive to salicylic acid (SA) but also PHYTOALEXIN DEFICIENT3 (PAD3), an enzyme responsible for the production …

   This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6513650.

   Phosphorus is an essential macro-nutrient and its availability to plants is known to modulate their immunity via the Phosphate Starvation Response (PSR). This was shown in Castrillo et al. 20171, where the authors demonstrated that an Arabidopsis thaliana (hereafter Arabidopsis) phr1 phl1 double mutant unable to deploy the PSR showed, under inorganic phosphate (Pi) limiting conditions, enhanced immunity levels transcriptionally and enhanced resistance to two biotrophic leaf pathogens. Genes highlighted in the transcriptome analysis notably include several transcription factors responsive to salicylic acid (SA) but also PHYTOALEXIN DEFICIENT3 (PAD3), an enzyme responsible for the production of the antimicrobial compound camalexin.  A recent study2 in rice showed that a high Pi treatment (2 mM) and subsequent Pi accumulation enhanced susceptibility to the blast fungus Magnaporthe oryzae. It was also shown in Dindas et al. 20213 that perception of microbial elicitors by plasma-membrane localized receptors directly inhibits Pi importer activity via phosphorylation. The PSR and plant immunity are undoubtedly linked and further investigations are crucial to understand which pathways are affected by the PSR, and in what way.  

   In this study, Val-Torregrosa and colleagues4 took a detailed look at how Pi influences the interaction between Arabidopsis and the necrotrophic fungus Plectosphaerella cucumerina. They utilized a Pi supply gradient and Pi hyper-accumulator mutants (miR827-OE and nla-1) to test whether and how P. cucumerina capacity to elicit disease on Arabidopsis leaves is altered. The main findings are that the nla-1 mutation (or miR827-OE) results in hyperaccumulation of Pi, enhanced resistance to P. cucumerina and higher levels of SA, jasmonic acid (JA), callose deposition and camalexin. Also, Pi gradient experiments showed that callose and camalexin production capacities increase with available Pi, an effect further compounded by nla-1. The key message the work puts forward is that the overaccumulation of Pi due to nla-1 causes enhanced immunity.

   The study is timely, in a research area enjoying a growing interest and it presents coherent findings made using appropriate methodologies. Here, we would like to suggest i) additional experiments to better clarify the authors' observations and perhaps strengthen the claim that NLA is a negative regulator of plant immunity via enhanced Pi accumulation and ii) manuscript improvements that would help clarify how the authors' present their work.

    

   Experimental consolidation

   1.     It was demonstrated that, compared to WT, nla-1 and miR827-OE plants are more resistant to P. cucumerina infection (Fig. 1), that nla-1 plants accumulate more camalexin and callose (Fig. 2 and Fig. 5) and that in WT plants, high Pi results in enhanced production of both camalexin and callose (Fig. 2 and Fig. 5). The missing, critical experiment is a disease assessment on a Pi gradient. One would predict that high Pi fertilization results in enhanced resistance towards P. cucumerina in WT and that could likely be further potentiated in nla-1. Conversely, low Pi fertilization might lead to severe disease in both WT and nla-1. Since in planta Pi accumulation is dependent on the Pi concentration provided to the plants (SFig. 4), a low Pi treatment (50 µM) would allow the authors to partially rule out a plausible "autoimmunity" phenotype due to the nla-1 mutation. In other words, if nla-1 enhances immunity via Pi accumulation, the WT and nla-1 might have comparable disease severity under the 50 µM Pi treatment because WT and nla-1 plants show comparable levels of accumulated Pi at 50 µM (SFig. 4). The authors have a working system in which both visible disease symptoms and fungal biomass are affected by the nla-1 mutation (Fig. 1 and SFig. 3). We encourage the authors to perform a Pi gradient experiment: on agar plates as they did for determining callose deposition and camalexin levels or alternatively by supplementing potting soil with liquid media containing the Pi gradient employed.  

    

   2.     Next, we would like to point out the possibility that the nla-1 mutation or miR827-OE causes a generally high immune activation, potentially "autoimmunity", independently of the Pi hyper accumulation. Hallmarks of autoimmunity can include constitutively high levels of SA and JA and associated marker genes, dwarfed plants, mild leaf chlorosis, curled leaves, spontaneous callose deposition and, occasionally, also spontaneous cell-death. The authors observed — under pathogen-free conditions — constitutively higher SA & JA levels. It was noted that in three-week-old plants "no phenotypic differences were observed between nla, miR827 OE and wild-type plants", yet "At a later developmental stage, however, miR827 OE plants displayed a reduced size compared with wild-type plants". Additionally, the nla leaves presented in Fig. S3 clearly display a lighter green pigmentation compared to WT leaves, a difference also apparent to a minor extent between miR827-OE and WT in Fig. S2. It could be that this is a simple light effect during photography. Otherwise, could it be leaf chlorosis? We would like to mention the fact that at 2 mM Pi, spontaneous callose deposition occurs in absence of P. cucumerina in both WT and nla (Fig. 2). Could 2 mM Pi fertilization be an osmotic stress that activates immune pathways, akin to salt stress5,6? We suggest that the authors perform a more detailed analysis of the general immune status of the plants, for instance, gene expression levels of ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4) as well as MAPK3/6 phosphorylation levels. This would clarify whether high Pi (2 mM fertilization or as a consequence of nla-1) causes a general autoimmunity or somehow only increases constitutive SA, JA and callose accumulation.

    

   We would also like to propose more ambitious experiments that would be interesting to at least mention in the discussion.

   3.     Ubiquitin E3 ligases confer substrate specificity for proteasomal degradation. Have the authors investigated, or is it known, whether there are close paralogs of NLA in Arabidopsis that could be part of the PSR or involved in immunity? Looking at and experimenting with such sequence diversification might be informative to better frame the role(s) of NLA.

    

   4.     It would be interesting to investigate whether there is a connection between JA signalling and the PSR. To this end, it might be useful to employ SA signalling mutants (e.g. npr1-1). Have the authors considered performing Methyl-jasmonate treatments (as they did for elicitors and SA) to test for induction of miR827prom::GUS and Pi import? Callose accumulation is known to be induced by SA-related outputs but is this also true for JA, which plays a critical part in Arabidopsis-necrotroph infections? What is the pathogen infection phenotype and Pi accumulation status in the nla-1 npr1-1 double mutant?

    

   Suggestions on the manuscript

   Finally, we suggest that the manuscript would greatly benefit if a number of important points were addressed in the text.

   1.     It is of importance that the authors justify the rationale behind the chosen Pi concentrations for the Pi gradient experiment, notably the higher end of the gradient (2 mM). Is it because this concentration was employed on rice in Campos-Soriano et al. 20202? Is it because, as the authors state, "less is known about adaptive mechanisms to Pi excess condition" at the end of the second introductory paragraph? Generally, what do the authors mean by "Pi excess"? Concentrations not encountered by plants in natural environments? Hyper-accumulation due to mutations, such as nla-1 in Arabidopsis? Please clarify.

    

   2.     In the third paragraph of the introduction, "Under low Pi conditions, the PHR1 transcription factor target genes involved in JA- and/or SA-dependent pathways for suppression of immune responses (Castrillo et al. 2017)." This statement is an overinterpretation and not what Castrillo and colleagues have shown. In Castrillo et al. 2017 Fig. 4, the double mutant phr1 phl1 shows higher immune activation under low Pi conditions, they did not demonstrate that PHR1 directly targets immunity genes. Please rephrase.

    

   3.     The fourth paragraph is an overview of how plant immunity was understood in the recent past. We would argue that the classical ETI-PTI dichotomy is now outdated7–9. Additionally, the authors went through the effort of introducing ETI and R genes despite the fact that they do not include these immune mechanisms in their analyses. We suggest that the introduction of plant immunity is reduced to what is essential to understand the authors' experiments. However, if the authors take on board the deeper characterization of the plausible "autoimmunity" in nla and look at ETI markers, they should of course present to readers the appropriate knowledge to understand the experiments.

    

   4.     The titles of the Results subsections are not consistently informative. For instance: "Callose accumulation in nla plants in response to P.cucumerina infection" does not convey that nla plants produce more callose than WT plants. Giving the Results subsections more informative titles would aid the reader in navigating through the manuscript.

    

   References

   1.         Castrillo, G. et al. Root microbiota drive direct integration of phosphate stress and immunity. Nature 543, 513–518 (2017).

   2.         Campos-Soriano, L. et al. Phosphate excess increases susceptibility to pathogen infection in rice. Mol. Plant Pathol. 21, 555–570 (2020).

   3.         Dindas, J. et al. Direct inhibition of phosphate transport by immune signaling in Arabidopsis. Curr. Biol. 1–8 (2021). doi:10.1016/j.cub.2021.11.063

   4.         Val-Torregrosa, B., Bundó, M., Chiou, T.-J., Flors, V. & Segundo, B. S. NITROGEN LIMITATION ADAPTATION functions as a negative regulator of Arabidopsis immunity. (2021).

   5.         Ariga, H. et al. NLR locus-mediated trade-off between abiotic and biotic stress adaptation in Arabidopsis. Nat. Plants 3, 17072 (2017).

   6.         Uchida, K. et al. MAP KINASE PHOSPHATASE1 promotes osmotolerance by suppressing PHYTOALEXIN DEFICIENT4-independent immunity. Plant Physiol. 1–11 (2022). doi:10.1093/plphys/kiac131

   7.         Yuan, M. et al. Pattern-recognition receptors are required for NLR-mediated plant immunity. Nature 592, 105–109 (2021).

   8.         Ngou, B. P. M., Ahn, H. K., Ding, P. & Jones, J. D. G. Mutual potentiation of plant immunity by cell-surface and intracellular receptors. Nature 592, 110–115 (2021).

   9.         Pruitt, R. N. et al. The EDS1–PAD4–ADR1 node mediates Arabidopsis pattern-triggered immunity. Nature 598, 495–499 (2021).

    

   Charles Uhlmann and the Plant Microbe Interactions PhD journal club at MPIPZ, Cologne 

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   Version published to 10.1101/2021.12.09.471910v1 on bioRxiv

   Dec 10, 2021