Transcriptome and proteome analysis reveal new insight into proximal and distal responses of wheat to foliar...
4
X. translucens infection activates a complex defense response at the site of infection
The first phase of the PTI response relies on the recognition of pathogen-derived molecules by pattern recognition receptors (PRRs). Transcriptomic data showed that a large number of receptor kinases were upregulated at the infected site (106 DEGs). Among these, we identified FLS2 and EFR, which are well-characterized leucine-rich repeat receptors that recognize bacterial flagellin and EF-Tu elongation factor, respectively. In addition, the upregulation of some receptor-like cytoplasmic kinases (RLCKs), such as Brassinosteroid signaling kinase 1 (BSK1) and PTI Compromised Receptor-like Cytoplasmic Kinase 1 (PCRK1), may contribute to PTI in response to X. translucens.
Two putative LysM-containing receptor-like kinase homologs of Arabidopsis LYK5 and CERK1, essential in the perception and signal transduction of the fungal chitin oligosaccharide elicitor, were upregulated by bacterial infection. In addition to its function in anti-fungal immunity, CERK1 is also involved in the perception of bacterial peptidoglycans33 and is required for resistance to bacterial infection34. These findings suggest the presence of an overlapping receptor system in wheat, as previously suggested in rice35. The recognition of different microbial ligands may contribute to a more rapid achievement of the signaling threshold needed for PTI activation.
There is increasing evidence that other receptor kinases, some of which were upregulated, act as additional plant immune receptors. Of these, several plant lectin and cysteine receptor kinases (CRKs) are upregulated upon perception of flagellin and act coordinately to enhance plant immune responses36,37,38. Expert manual data analysis also revealed upregulation of the Clavata 2 (CLV2) transcript. Very recent data indicate that CLV2 is implicated in plant immunity39. Six DEGs encoding leucine-rich repeat receptor kinases involved in the perception of sulfated peptide PSY1 were strongly overexpressed at the site of infection. The activation of these receptors leads to the downregulation of SA-related responses and upregulation of JA signaling after infection by a biotrophic pathogen. PSY1 also promotes plant growth40. Our data confirm that PSY1 signaling is important for the integration of plant growth with defense responses.
Finally, several wall-associated receptor kinases (WAKs) were upregulated. One of these, WAK1, recognizes oligogalacturonides (OGs), which are degradation products of the plant cell wall released upon pathogen infection, and triggers immune responses41.
Our experiments suggest that the blue-light photoreceptors PHOT1 and CRY1 were downregulated in response to infection. Both genes are involved in blue light-induced stomatal opening and their downregulation may lead to stomatal closure to restrict bacterial invasion. Upregulation of three members of the Plant U-box family of ubiquitin E3 ligases (PUB22, PUB23 and PUB12), which are involved in proteasomal degradation of PRR complexes42, reveals an additional layer of complexity. Protein turnover may contribute to tight control of PRR abundance to avoid inappropriate activation.
Overall, these data demonstrate the amplitude and complexity of the PRR response to a single microbial species. The accumulation of plasma membrane receptors may lead to stronger and faster immune responses against subsequent pathogen attack (data not shown).
Diverse signaling pathways are stimulated at the infection site
Signaling events observed after pathogen recognition are mediated by ion fluxes across the plasma membrane, such as an influx of Ca2+ into the cytosol, a burst of ROS, and the activation of protein kinase cascades. Genes encoding calmodulin-binding proteins, calcineurin proteins, calcium ATPase, and glutamate receptor-like channels (GLURs) were upregulated at the site of infection. These proteins constitute a network that can induce and maintain intracellular Ca2+ levels to trigger signaling pathways. For example, GLURs were proposed to act as amino acid (AA)-gated calcium channels to perceive changes in the apoplastic AA concentrations resulting either from cell damage or from PAMP-induced exocytosis43. Our data confirm that GLURs can contribute to PTI activation in parallel with PRRs (data not shown).
Upregulation of some calcium-dependent protein kinases (CDPKs) was also observed. CDPKs were proposed to function in multiple plant signal transduction pathways downstream of Ca2+ cytoplasmic elevation. CPK1 can phosphorylate phenylalanine ammonia lyase (PAL), a key enzyme in pathogen defense, while CPK3 functions in guard cell ion channel regulation.
MAPK cascades are known to be implicated in plant immunity. In this study, we observed the upregulation of EDR1, MAPKKK5, MAPKKK15, MAPKKK3, MAPKK2, MAPKK5, MPK9, MPK3, and MPK4. EDR1 is a MAPKKK serine/threonine-protein kinase that is involved in the regulation of a MAP kinase cascade (probably including MPK3 and MPK6) that negatively regulates SA-dependent defense responses, ABA signaling, and ET-induced senescence44.
PTI can be attenuated by effector molecules secreted into plant cells by microbial pathogens45. Plants have evolved a class of immune receptors, encoded by disease resistance (R)-genes that recognize the presence or activity of effectors and induce the effector-triggered immunity (ETI) response. In this study, 149 DEGs that encoded disease resistance genes, e.g. RPM1 and RPS2, were differentially regulated at the site of infection. Although ETI constitutes a stronger immune response that is often associated with the hypersensitive reaction (HR), a form of programmed death of plant cells (PCD) at infection sites, the ETI and PTI gene expression signatures were largely similar. Metacaspase AMC1, a positive regulator of PCD46, and four proteins involved in suppression of cell death (Bax inhibitor 1, ATDAD1, CAD1, and BONZAI 3) were upregulated concurrently. These data indicate that plant cell death is simultaneously stimulated and repressed, maintaining cellular homeostasis. This may explain the lack of symptoms at the pathogen entry site 24 hpi.
Chloroplasts play a central role against X. translucens attack
Some studies suggest that photosynthesis is essential for the defense response47. Our transcriptomic and proteomic data showed that X. translucens infection had a substantial effect on components of the photosynthetic apparatus and on chloroplast biogenesis in the infected leaf. Indeed, a very large number of genes and proteins related to photosystem II, chlorophyll and carotenoid biosynthesis were significantly downregulated in treated leaves compared with control leaves. These included transcripts encoding the FLU and GUN4 Arabidopsis homologs required for tetrapyrrole biosynthesis regulation. Moreover, the Calcium-sensing protein (CAS) was deregulated. These latter proteins are involved in retrograde signaling and their impairment leads to ROS accumulation in the chloroplast48. Finally, the transcription factor NF-YB, which regulates the expression of nuclear-encoded chloroplast-targeted genes and the normal development of chloroplasts49, was affected in treated leaves at both the RNA and protein levels.
Transcriptomic data showed that some Calvin cycle genes were downregulated, while proteomic analysis showed accumulation of four key enzymes of the cycle in infected leaves. A similar lack of correlation between levels of mRNA and protein involved in photosynthesis was reported by Gohre et al.50. These data suggest that the plant attempts to maintain carbon assimilation stable during pathogenic attack.
In infected leaves, the expression levels of some genes and proteins that have important roles in chloroplast sulfate assimilation, including sirohydrochlorin ferrochelatase B (SirB), ATP-sulfurylase, and cysteine synthases, were significantly decreased. Cysteine acts as a precursor or donor of reduced S for a range of S-compounds, such as methionine (Met), and glutathione (GSH). Several enzymes involved in Met salvage, a Met recycling pathway, were upregulated. Met regeneration may play an important role in sustaining the production of ET in infected tissues (see below).
Siroheme is a cofactor of ferredoxin-nitrite reductase (NiR), and, accordingly, the level of NiR was significantly downregulated in infected leaves. At the transcriptomic level, two nitrate reductases were downregulated and some DEGs corresponding to ammonium transporter (AMT2) genes were strongly induced in leaves. By contrast, in the roots, proteomic data showed that NiR, AMT2, and one high-affinity nitrate transporter were upregulated.
Transcription of chloroplastic precursors of the glutamine synthase GS2 and the ferredoxin-dependent glutamate aminotransferase (Fd-GOGAT), involved in primary nitrogen assimilation, also decreased, while the genes encoding cytoplasmic GS and NADH-GOGAT were strongly induced. Finally, the observed increase in glutamate dehydrogenase transcripts indicated that glutamate was used as a source of energy and carbon in infected leaves. Overall, these data indicate that 1) the chloroplast is a strategic battlefield during pathogen attack and, confirming recent research, is also a key component of the plant immune response51; 2) remobilization is the main source of nitrogen in the infected leaf, whereas the root is involved in primary N assimilation.
ROS and redox regulation are important contributors to the wheat response to X. translucens
The production of ROS is one of the first responses that occurs after pathogen recognition52, 53. Accordingly, our datasets showed the upregulation of NADPH/respiratory burst oxidase protein D (RbohD) and of extracellular peroxidases, major regulators of the apoplastic oxidative burst around the infection site that are also involved in cross-linking cell wall components54, 55.
In infected leaves, 35 of 45 DEGs encoding ascorbate peroxidases (APXs) were strongly upregulated. APX enzymes play a key role in catalyzing the conversion of H2O2 to H2O, using ascorbate or GSH as a specific electron donor. Conversely, at the protein level, there was abundance of only one APX, while levels of three APX proteins decreased. Two of the three downregulated proteins were stromal and thylakoid membrane-bound APXs: sAPX and tAPX, respectively. These key enzymes are involved in H2O2 scavenging in chloroplasts. tAPX reduction enhances nitric oxide-induced cell death56 and activates defense responses57. Our findings support the involvement of tAPX in the response to pathogens, probably through slight changes in the H2O2 concentration in chloroplasts.
Several chloroplast thioredoxins were downregulated in leaves at both the RNA and protein levels. The major function of these proteins is to reduce the disulfide bonds of their substrate proteins and maintain cellular redox homeostasis. Glutathione S-transferases (GSTs) were mostly upregulated at proteomic and transcriptomic levels.
Proteomic results indicated that some enzymes involved in ROS detoxification and cell redox homeostasis, such as APXs, GSTs, and thioredoxins, also accumulated in the roots of infected plants. Some SODs were upregulated at the transcriptomic and proteomic levels. The response of peroxidases was less evident, because nine of the ten peroxidases identified were downregulated at the transcriptomic level, while proteomic analysis showed the accumulation of five peroxidases among the seven that were identified. We can speculate that the downstream mediators of foliar pathogen attack induce mild oxidative stress in roots, enhancing the accumulation of proteins involved in ROS scavenging.
We studied the accumulation of oxidative modified polypeptides by conducting an immunoblot analysis of carbonylated proteins. Protein carbonylation, which is irreversible, is one of the most harmful oxidative protein modifications and is considered a major hallmark of oxidative damage58. The extent of protein carbonylation was lower both in leaves and in the roots of plants exposed to X. translucens than in control samples (Fig. 5). This result indicates that the antioxidant defense system was able to cope with the oxidative stress induced by bacterial infection at 24 hpi, even in infected leaves.
5
Protein carbonylation profiles of wheat plants after Xanthomonas infection: (A) leaves protein stain (left), anti-DNP (2,4-dinitrophenol) immunoassay (right); (B) roots protein stain (left), anti-DNP (2,4-dinitrophenol) immunoassay (right); (C) relative protein carbonylation values (referred to the control sample) expressed as the carbonylation index, after normalization for protein amounts. Data (means ± SD, n = 3) were subjected to one-way analysis of variance (ANOVA). The asterisks indicate significant differences at the 5% level using Tukey’s test.
X. translucens infection induces metabolic changes in proximal and distal plant tissues
The decrease in photosynthesis leads to a metabolic shift of infected tissues from source to sink. The induction of cell wall invertases (cw-Inv) and hexose and sucrose transporters is considered to be the primary cause for the formation of a sink at the infection site59. Of 22 DEGs encoding transcripts for vacuolar and apoplastic invertases, 14 were upregulated, while at the proteomic level one cw-Inv protein was downregulated. Sonnewald et al. demonstrated that X. campestris pv. vesicatoria suppressed cw-Inv activity in a T3SS-dependent manner60. Invertases cleave sucrose into glucose and fructose, thereby preventing sucrose export from infected cells. Furthermore, 25/43 DEGs coding for sugar transporters were overexpressed. One of these was a transcript encoding a STP13 homolog. It was recently demonstrated that STP13 reduces sugar content in the Arabidopsis apoplast, resulting in limited bacterial proliferation61.
Hexokinase (HXK), the best investigated glucose sensor, can show the energy status of the cell and induce subsequent signaling cascades to modulate cellular metabolism in response to carbon status62. Increasing evidence indicates that hexoses help defense responses59. Some HXKs accumulated in infected leaves, possibly contributing to the generation of ROS and the activation of PR genes63. Several other sugar kinases, such as galactokinase1 (GAL1) and arabinose kinase 1 (ARA1), were overexpressed in infected leaves; however, it is not yet clear whether these enzymes have signaling functions similar to that proposed for HXK.
This study showed that AA anabolic and catabolic pathways were also modified in infected leaves. Regulation of AA content and transport is critical for plant adaptation to carbon and nitrogen status, development, and defense64. Several enzymes involved in aspartate, alanine, proline, asparagine, arginine, methionine, lysine, phenylalanine, tyrosine, and tryptophan biosynthesis were induced at the site of X. translucens infection, as shown by transcriptomic and proteomic analysis.
Glutamine, asparagine, arginine, and proline may be involved in N recycling, remobilization, and translocation in infected leaves. Accordingly, some DEGs coding for an asparagine synthetase, ASN1, whose expression responds to the level of sugar in the cell65, were overexpressed. Conversely, DEGs coding for asparaginase were downregulated. Many molecules involved in plant defense are derived from AA, such as ET from methionine and phytoalexin, anthocyanin, and SA from phenylalanine. Some DEGs encoding AA metabolism enzymes that are involved in plant immunity were overexpressed. Of these, ornithine-d-aminotransferase and aspartate oxidase were previously shown to contribute to pathogen-induced ROS production and HR66.
Finally, some DEGs corresponding to AGD2-like defense response protein 1 (ALD1) homologs (involved in lysine degradation) were strongly overexpressed. ALD1 transcript levels rise both locally and systemically in pathogen-inoculated Arabidopsis67. The lysine catabolite pipecolic acid (Pip) plays an important role in defense amplification and priming that allows plants to acquire immunity at the systemic level68. Recently, it was proposed that FMO1, whose transcripts were upregulated in our experiments, acts as a critical mediator for Pip-activated responses69. We speculate that Pip could function as a mobile element in shoot-root communication during foliar attack by X. translucens.
Foliar infection induced a metabolic change in the roots. Briefly, a SnRK1 kinase that was involved in the regulation of nitrogen and in the metabolism of sugar, and some genes and proteins related to AA metabolism, were upregulated at the RNA and protein levels. Some mitochondrial membrane metabolite transporters and some components of the respiratory chain complex were also upregulated. Components of the mitochondrial permeability transition alter their abundance during the plant basal defense response70. These data suggest that mitochondria in roots respond to aboveground attack and may supply energy for metabolic changes in both roots and leaves.
Overall, these data indicate the necessity of maintaining energy and solute homeostasis for cell protection during pathogen progression both in proximal and distal plant tissues.
Secondary metabolism and defense-related proteins
Consistent with previous research, our datasets showed that key enzymes of the phenylpropanoid pathway were triggered during pathogen attack71, 72. The cell wall is one of the most important barriers against pathogens, and lignification is important in plant defense73. This lignification was very clear at the transcriptomic and proteomic levels in the present study, as shown in Supplementary Figure 3. Some of the key enzymes involved in the biosynthesis and polymerization of lignin (such as caffeoyl CoA O-methyltransferase, 4-coumarate-CoA ligase, cynnamoyl-CoA dehydrogenase, and laccase) accumulated in the leaves and roots of infected plants. The upregulation of four dirigent-like proteins, which are part of the machinery that builds extracellular lignin-based structures74, was also observed in both the leaves and roots. Genes and proteins involved in isoprenoid biosynthesis were downregulated in both the leaves and roots, while the biosynthesis of flavonoids appeared to be upregulated. Lastly, enzymes involved in alkaloid biosynthesis, such as strictosidine synthase and tropinone reductase, were upregulated in the roots.
We observed the alteration of several enzymes involved in cell wall remodeling after X. translucens invasion in leaves, including polygalacturonases, alpha- and beta-glucosidases, alpha-fucosidase, beta-D-xylosidase, and pectin acetylesterase. Cell wall remodeling may affect intracellular signal transduction and the cell defense response at the site of pathogen attack. In infected leaves, we also observed accumulation of an auxin-inducible expansin. Expansins, which mediate long-term extension of the cell wall, can render the plant cell wall vulnerable, creating an opportunity for pathogen attack. Xylan is the major hemicellulose polymer in cereals. Infected leaves can counteract microbial endoxylanases by accumulating xylanase inhibitors, as shown by our RNAseq and proteomic data. In the roots, the accumulation of proteins involved in cell wall degradation (one endo-beta-mannosidase, one β-1,4-glucanase, and one polygalacturonase) suggests a change in root development upon foliar attack.
In conclusion, foliar attack by X. translucens increases secondary metabolite concentrations in roots to a similar extent as observed locally in leaves. Similar results were observed in plants attacked by leaf herbivores3, 4. It was proposed that synthesis of defense proteins in roots upon foliar infection may allow plants to continue to protect themselves, even when large parts of the aboveground components of the plant are destroyed. Moreover, the fact that roots allow access to nitrogen and phosphate in the soil may mean that the production of certain secondary metabolites in roots is advantageous4. The upregulation of nitrogen transporters and nitrate reductase in the roots in our experiments supports this hypothesis. Several studies showed that secondary metabolites and cysteine proteases that were upregulated in roots were synthesized in the roots and transported to the shoot to provide resistance to foliar attack75.
Comments