SNARE SYP132 mediates divergent traffic of plasma membrane H+-ATPase AHA1 and antimicrobial PR1 during bacterial pathogenesis

The vesicle trafficking SYNTAXIN OF PLANTS132 (SYP132) drives hormone-regulated endocytic traffic to suppress the density and function of plasma membrane (PM) H⁺-ATPases. In response to bacterial pathogens, it also promotes secretory traffic of antimicrobial pathogenesis-related (PR) proteins. These seemingly opposite actions of SYP132 raise questions about the mechanistic connections between the two, likely independent, membrane trafficking pathways intersecting plant growth and immunity. To study SYP132 and associated trafficking of PM H⁺-ATPase 1 (AHA1) and PATHOGENESIS-RELATED PROTEIN1 (PR1) during pathogenesis, we used the virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) bacteria for infection of Arabidopsis (Arabidopsis thaliana) plants. SYP132 overexpression suppressed bacterial infection in plants through the stomatal route. However, bacterial infection was enhanced when bacteria were infiltrated into leaf tissue to bypass stomatal defenses. Tracking time-dependent changes in native AHA1 and SYP132 abundance, cellular distribution, and function, we discovered that bacterial pathogen infection triggers AHA1 and SYP132 internalization from the plasma membrane. AHA1 bound to SYP132 through its regulatory SNARE Habc domain, and these interactions affected PM H⁺-ATPase traffic. Remarkably, using the Arabidopsis aha1 mutant, we discovered that AHA1 is essential for moderating SYP132 abundance and associated secretion of PR1 at the plasma membrane for pathogen defense. Thus, we show that during pathogenesis SYP132 coordinates AHA1 with opposing effects on the traffic of AHA1 and PR1.

Coordination between SNARE SYP132 and plasma membrane H⁺-ATPase AHA1 moderates SNARE abundance during pathogenesis with opposing effects on trafficking of AHA1 and antimicrobial pathogenesis-related protein 1.     

Regulation of disease-responsive genes mediated by epigenetic factors: interaction of Arabidopsis-Pseudomonas

Genes in eukaryotic organisms function within the context of chromatin, and the mechanisms that modulate the structure of chromatin are defined as epigenetic. In Arabidopsis, pathogen infection induces the expression of at least one histone deacetylase, suggesting that histone acetylation/deacetylation has an important role in the pathogenic response in plants. How/whether histone methylation affects gene response to pathogen infection is unknown. To gain a better understanding of the epigenetic mechanisms regulating the interaction between Pseudomonas syringae and Arabidopsis thaliana, we analysed three different Arabidopsis ash1-related (absent, small or homeotic discs 1) mutants. We found that the loss of function of ASHH2 and ASHR1 resulted in faster hypersensitive responses (HRs) to both mutant (hrpA) and pathogenic (DC3000) strains of P. syringae, whereas control (Col-0) and ashr3 mutants appeared to be more resistant to the infection after 2 days. Furthermore, we showed that, in the ashr3 background, the PR1 gene (PATHOGENESIS-RELATED GENE 1) displayed the highest expression levels on infection with DC3000, correlating with increased resistance against this pathogen. Our results show that, in both the ashr1 and ashh2 backgrounds, the histone H3 lysine 4 dimethylation (H3K4me2) levels decreased at the promoter region of PR1 on infection with the DC3000 strain, suggesting that an epigenetically regulated PR1 expression is involved in the plant defence. Our results suggest that histone methylation in response to pathogen infection may be a critical component in the signalling and defence processes occurring between plants and microbes.     

The Role of Autophagy in Chloroplast Degradation and Chlorophagy in Immune Defenses during Pst DC3000 (AvrRps4) Infection

Background

Chlorosis of leaf tissue normally observed during pathogen infection may result from the degradation of chloroplasts. There is a growing evidence to suggest that the chloroplast plays a significant role during pathogen infection. Although most degradation of the organelles and cellular structures in plants is mediated by autophagy, its role in chloroplast catabolism during pathogen infection is largely unknown.

Results

In this study, we investigated the function of autophagy in chloroplast degradation during avirulent Pst DC3000 (AvrRps4) infection. We examined the expression of defensive marker genes and suppression of bacterial growth using the electrolyte leakage assay in normal light (N) and low light (L) growing environments of wild-type and atg5-1 plants during pathogen treatment. Stroma-targeted GFP proteins (CT-GFP) were observed with LysoTracker Red (LTR) staining of autophagosome-like structures in the vacuole. The results showed that Arabidopsis expressed a significant number of small GFP-labeled bodies when infected with avirulent Pst DC3000 (AvrRps4). While barely detectable, there were small GFP-labeled bodies in plants with the CT-GFP expressing atg5-1 mutation. The results showed that chloroplast degradation depends on autophagy and this may play an important role in inhibiting pathogen growth.

Conclusion

Autophagy plays a role in chloroplast degradation in Arabidopsis during avirulent Pst DC3000 (AvrRps4) infection. Autophagy dependent chloroplast degradation may be the primary source of reactive oxygen species (ROS) as well as the pathogen-response signaling molecules that induce the defense response.     

The impact of cytokinin on jasmonate-salicylate antagonism in Arabidopsis immunity against infection with Pst DC3000

Cytokinin has long been shown to be an essential modulator of growth and development in plants. However, its implications in plant immunity have only recently been realized. The interaction between jasmonate and salicylate pathways is regarded as a central backbone of plant immune defense. However, the effect of cytokinin on the jasmonate and salicylate mediated balance in plant immunity is still not known. Here, we analyze the impact of cytokinin on the jasmonate-salicylate antagonism in Arabidopsis immunity regarding infection with hemibiotrophic pathogen Pseudomonas syringae pv tomato DC3000 (Pst DC3000). Systems biology analysis of a refined hormone immune pathway model provides insights into the impact of cytokinin on the balance between jasmonate and salicylic acid pathways in Arabidopsis. Targeted experiments validate model simulations monitoring bacterial growth in wild type plants as well as in jasmonate pathway mutants. An integrated analysis shows that CK promotes the SA pathway of plant immunity and does not promote JA-mediated Arabidopsis susceptibility against infection with Pst DC3000. Finally, we discuss these results in the context of an emerging model of auxin-cytokinin antagonism in plant immunity.     

An Extracellular Subtilase Switch for Immune Priming in Arabidopsis

In higher eukaryotes, induced resistance associates with acquisition of a priming state of the cells for a more effective activation of innate immunity; however, the nature of the components for mounting this type of immunological memory is not well known. We identified an extracellular subtilase from Arabidopsis, SBT3.3, the overexpression of which enhances innate immune responses while the loss of function compromises them. SBT3.3 expression initiates a durable autoinduction mechanism that promotes chromatin remodeling and activates a salicylic acid(SA)-dependent mechanism of priming of defense genes for amplified response. Moreover, SBT3.3 expression-sensitized plants for enhanced expression of the OXI1 kinase gene and activation of MAP kinases following pathogen attack, providing additional clues for the regulation of immune priming by SBT3.3. Conversely, in sbt3.3 mutant plants pathogen-mediated induction of SA-related defense gene expression is drastically reduced and activation of MAP kinases inhibited. Moreover, chromatin remodeling of defense-related genes normally associated with activation of an immune priming response appear inhibited in sbt3.3 plants, further indicating the importance of the extracellular SBT3.3 subtilase in the establishment of immune priming. Our results also point to an epigenetic control in the regulation of plant immunity, since SBT3.3 is up-regulated and priming activated when epigenetic control is impeded. SBT3.3 represents a new regulator of primed immunity.     

Unlocking the Arabidopsis epigenome

The patterns of DNA methylation, referred to as the “methylome”, must be faithfully propagated for proper development of plants and mammals. However, it has been unclear to which extent transgenerational epigenetic inheritance will be affected after DNA methylation distribution has been altered. Recently, three reports have addressed this issue in the model plant Arabidopsis thaliana. Here we revisit the results of these experiments addressing the stability of epigenetic inheritance within two populations of epigenetic recombinant inbred lines (epiRILs), in which mosaic epigenomes were subjected to inbreeding for multiple generations. The manner in which the epigenetic variation was induced differed between the two populations, one by adversely affecting chromatin remodeling and the second by impairing the maintenance of DNA methylation, yet the comparison of the results provides a broader view of transgenerational epigenetic inheritance that may find parallels in other organisms.     

Sucrose-mediated priming of plant defense responses and broad-spectrum disease resistance by overexpression of the maize pathogenesis-related PRms protein in rice plants

Expression of pathogenesis-related (PR) genes is part of the plant's natural defense response against pathogen attack. The PRms gene encodes a fungal-inducible PR protein from maize. Here, we demonstrate that expression of PRms in transgenic rice confers broad-spectrum protection against pathogens, including fungal (Magnaporthe oryzae, Fusarium verticillioides, and Helminthosporium oryzae) and bacterial (Erwinia chrysanthemi) pathogens. The PRms-mediated disease resistance in rice plants is associated with an enhanced capacity to express and activate the natural plant defense mechanisms. Thus, PRms rice plants display a basal level of expression of endogenous defense genes in the absence of the pathogen. PRms plants also exhibit stronger and quicker defense responses during pathogen infection. We also have found that sucrose accumulates at higher levels in leaves of PRms plants. Sucrose responsiveness of rice defense genes correlates with the pathogen-responsive priming of their expression in PRms rice plants. Moreover, pretreatment of rice plants with sucrose enhances resistance to M. oryzae infection. Together, these results support a sucrose-mediated priming of defense responses in PRms rice plants which results in broad-spectrum disease resistance.     

Priming for enhanced defence responses by specific inhibition of the Arabidopsis response to coronatine

The priming agent β-aminobutyric acid (BABA) is known to enhance Arabidopsis resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 by potentiating salicylic acid (SA) defence signalling, notably PR1 expression. The molecular mechanisms underlying this phenomenon remain unknown. A genome-wide microarray analysis of BABA priming during Pst DC3000 infection revealed direct and primed up-regulation of genes that are responsive to SA, the SA analogue benzothiadiazole and pathogens. In addition, BABA was found to inhibit the Arabidopsis response to the bacterial effector coronatine (COR). COR is known to promote bacterial virulence by inducing the jasmonic acid (JA) response to antagonize SA signalling activation. BABA specifically repressed the JA response induced by COR without affecting other plant JA responses. This repression was largely SA-independent, suggesting that it is not caused by negative cross-talk between SA and JA signalling cascades. Treatment with relatively high concentrations of purified COR counteracted BABA inhibition. Under these conditions, BABA failed to protect Arabidopsis against Pst DC3000. BABA did not induce priming and resistance in plants inoculated with a COR-deficient strain of Pst DC3000 or in the COR-insensitive mutant coi1-16. In addition, BABA blocked the COR-dependent re-opening of stomata during Pst DC3000 infection. Our data suggest that BABA primes for enhanced resistance to Pst DC3000 by interfering with the bacterial suppression of Arabidopsis SA-dependent defences. This study also suggests the existence of a signalling node that distinguishes COR from other JA responses.     

The epigenetic machinery controlling transgenerational systemic acquired resistance

Progeny from diseased Arabidopsis shows enhanced resistance, which is associated with priming of defense genes.¹ This transgenerational systemic acquired resistance (SAR) is effective against biotrophic pathogens, such as the downy mildew pathogen Hyaloperonospora arabidopsidis. In this study, we have examined mutants in RNA-directed DNA methylation (RdDM) for transgenerational SAR. Our analysis suggests that transgenerational SAR is regulated by the RdDM pathway and transmitted by hypomethylation at CpNpG sites.     

Bacteria‐type‐specific biparental immune priming in the pipefish Syngnathus typhle

The transfer of acquired and specific immunity against previously encountered bacteria from mothers to offspring boosts the immune response of the next generation and supports the development of a successful pathogen defense. While most studies claim that the transfer of immunity is a maternal trait, in the sex‐role‐reversed pipefish Syngnathus typhle, fathers nurse the embryos over a placenta‐like structure, which opens the door for additional paternal immune priming. We examined the potential and persistence of bacteria‐type‐specific parental immune priming in the pipefish S. typhle over maturation time using a fully reciprocal design with two different bacteria species (Vibrio spp. and Tenacibaculum maritimum). Our results suggest that S. typhle is able to specifically prime the next generation against prevalent local bacteria and to a limited extent even also against newly introduced bacteria species. Long‐term protection was thereby maintained only against prevailing Vibrio bacteria. Maternal and paternal transgenerational immune priming can complement each other, as they affect different pathways of the offspring immune system and come with distinct degree of specificity. The differential regulation of DNA‐methylation genes upon parental bacteria exposure in premature pipefish offspring indicates that epigenetic regulation processes are involved in transferring immune‐related information across generations. The identified trade‐offs between immune priming and reproduction determine TGIP as a costly trait, which might constrain the evolution of long‐lasting TGIP, if parental and offspring generations do not share the same parasite assembly.     

AvrBsT Acetylates Arabidopsis ACIP1, a Protein that Associates with Microtubules and Is Required for Immunity

Bacterial pathogens of plant and animals share a homologous group of virulence factors, referred to as the YopJ effector family, which are translocated by the type III secretion (T3S) system into host cells during infection. Recent work indicates that some of these effectors encode acetyltransferases that suppress host immunity. The YopJ-like protein AvrBsT is known to activate effector-triggered immunity (ETI) in Arabidopsis thaliana Pi-0 plants; however, the nature of its enzymatic activity and host target(s) has remained elusive. Here we report that AvrBsT possesses acetyltransferase activity and acetylates ACIP1 (for ACETYLATED INTERACTING PROTEIN1), an unknown protein from Arabidopsis. Genetic studies revealed that Arabidopsis ACIP family members are required for both pathogen-associated molecular pattern (PAMP)-triggered immunity and AvrBsT-triggered ETI during Pseudomonas syringae pathovar tomato DC3000 (Pst DC3000) infection. Microscopy studies revealed that ACIP1 is associated with punctae on the cell cortex and some of these punctae co-localize with microtubules. These structures were dramatically altered during infection. Pst DC3000 or Pst DC3000 AvrRpt2 infection triggered the formation of numerous, small ACIP1 punctae and rods. By contrast, Pst DC3000 AvrBsT infection primarily triggered the formation of large GFP-ACIP1 aggregates, in an acetyltransferase-dependent manner. Our data reveal that members of the ACIP family are new components of the defense machinery required for anti-bacterial immunity. They also suggest that AvrBsT-dependent acetylation in planta alters ACIP1''s defense function, which is linked to the activation of ETI.     

Riboflavin-induced Priming for Pathogen Defense in Arabidopsis thaliana

Riboflavin (vitamin B2) participates in a variety of redox processes that affect plant defense responses. Previously we have shown that riboflavin induces pathogen resistance in the absence of hypersensitive cell death (HCD) in plants. Herein, we report that riboflavin induces priming of defense responses in Arabidopsis thaliana toward infection by virulent Pseudomonas syringae pv. Tomato DC3000 (Pst). Induced resistance was mechanistically connected with the expression of defense response genes and cellular defense events, including H2O2 burst, HCD, and callose deposition in the plant. Riboflavin treatment and inoculation of plants with Pst were neither active but both synergized to induce defense responses. The priming process needed NPR1 (essential regulator of systemic acquired resistance) and maintenance of H2O2 burst but was independent of salicylic acid, jasmonic acid, ethylene, and abscisic acid. Our results suggest that the role of riboflavin in priming defenses is subject to a signaling process distinct from the known pathways of hormone signal transduction.     

Small RNAs, RNAi and the Inheritance of Gene Silencing in Caenorhabditis elegans

Invasive nucleic acids such as transposons and viruses usually exhibit aberrant characteristics, e.g., unpaired DNA or abnormal double-stranded RNA. Organisms employ a variety of strategies to defend themselves by distinguishing self and nonself substances and disabling these invasive nucleic acids. Furthermore, they have developed ways to remember this exposure to invaders and transmit the experience to their descendants. The mechanism underlying this inheritance has remained elusive. Recent research has shed light on the initiation and maintenance of RNA-mediated inherited gene silencing. Small regulatory RNAs play a variety of crucial roles in organisms, including gene regulation, developmental timing, antiviral defense, and genome integrity, via a process termed as RNA interference (RNAi). Recent research has revealed that small RNAs and the RNAi machinery are engaged in establishing and promoting transgenerational gene silencing. Small RNAs direct the RNAi and chromatin modification machinery to the cognate nucleic acids to regulate gene expression and epigenetic alterations. Notably, these acquired small RNAs and epigenetic changes persist and are transmitted from parents to offspring for multiple generations. Thus, RNAi is a vital determinant of the inheritance of gene silencing and acts as a driving force of evolution.