IDL6‐HAE/HSL2 impacts pectin degradation and resistance to Pseudomonas syringae pv tomato DC3000 in Arabidopsis leaves

Plant cell walls undergo dynamic structural and chemical changes during plant development and growth. Floral organ abscission and lateral root emergence are both accompanied by cell‐wall remodeling, which involves the INFLORESCENCE DEFICIENT IN ABSCISSION (IDA)‐derived peptide and its receptors, HAESA (HAE) and HAESA‐LIKE2 (HSL2). Plant cell walls also act as barriers against pathogenic invaders. Thus, the cell‐wall remodeling during plant development could have an influence on plant resistance to phytopathogens. Here, we identified IDA‐like 6 (IDL6), a gene that is prominently expressed in Arabidopsis leaves. IDL6 expression in Arabidopsis leaves is significantly upregulated when the plant is suffering from attacks of the bacterial Pseudomonas syringae pv. tomato (Pst) DC3000. IDL6 overexpression and knockdown lines respectively decrease and increase the Arabidopsis resistance to Pst DC3000, indicating that the gene promotes the Arabidopsis susceptibility to Pst DC3000. Moreover, IDL6 promotes the expression of a polygalacturonase (PG) gene, ADPG2, and increases PG activity in Arabidopsis leaves, which in turn reduces leaf pectin content and leaf robustness. ADPG2 overexpression restrains Arabidopsis resistance to Pst DC3000, whereas ADPG2 loss‐of‐function mutants increase the resistance to the bacterium. Pst DC3000 infection elevates the ADPG2 expression partially through HAE and HSL2. Taken together, our results suggest that IDL6‐HAE/HSL2 facilitates the ingress of Pst DC3000 by promoting pectin degradation in Arabidopsis leaves, and Pst DC3000 might enhance its infection by manipulating the IDL6‐HAE/HSL2‐ADPG2 signaling pathway.     

Brassinosteroids are involved in ethylene‐induced Pst DC3000 resistance in Nicotiana benthamiana

• Plant immunity is regulated by a huge phytohormone regulation network. Ethylene(ET) and brassinosteroids (BRs) play critical roles in plant response to biotic stress; however, the relationship between BR and ET in plant immunity is unclear.
• We used chemical treatments, genetic approaches and inoculation experiments to investigate the relationship between ET and BR in plant defense against Pst DC3000 in Nicotiana benthamiana.
• Foliar applications of ET and BR enhanced plant resistance to Pst DC3000 inoculation, while treatment with brassinazole (BRZ, a specific BR biosynthesis inhibitor) eliminated the ET induced plant resistance to Pst DC3000. Silencing of DWARF 4(DWF4, a key BR biosynthetic gene), BRASSINOSTEROID INSENSITIVE 1 (BRI1, aBR receptor) and BRASSINOSTEROID-SIGNALING KINASE 1 (BSK1, downstream of BRI1) also neutralised the ET‐induced plant resistance to Pst DC3000. ET can induce callose deposition and reactive oxygen species (ROS) accumulation to resistPst DC3000, BRZ‐treated and gene‐silenced were completely eliminate this response.
• Our results suggest BR is involved in ET‐induced plant resistance, the involvement of ET in plant resistance is possibly by the induction of callose deposition and ROS accumulation, in a BR‐dependent manner.

     

Distinct Pseudomonas type‐III effectors use a cleavable transit peptide to target chloroplasts

The pathogen Pseudomonas syringae requires a type‐III protein secretion system and the effector proteins it injects into plant cells for pathogenesis. The primary role for P. syringae type‐III effectors is the suppression of plant immunity. The P. syringae pv. tomato DC3000 HopK1 type‐III effector was known to suppress the hypersensitive response (HR), a programmed cell death response associated with effector‐triggered immunity. Here we show that DC3000 hopK1 mutants are reduced in their ability to grow in Arabidopsis, and produce reduced disease symptoms. Arabidopsis transgenically expressing HopK1 are reduced in PAMP‐triggered immune responses compared with wild‐type plants. An N‐terminal region of HopK1 shares similarity with the corresponding region in the well‐studied type‐III effector AvrRps4; however, their C‐terminal regions are dissimilar, indicating that they have different effector activities. HopK1 is processed in planta at the same processing site found in AvrRps4. The processed forms of HopK1 and AvrRps4 are chloroplast localized, indicating that the shared N‐terminal regions of these type‐III effectors represent a chloroplast transit peptide. The HopK1 contribution to virulence and the ability of HopK1 and AvrRps4 to suppress immunity required their respective transit peptides, but the AvrRps4‐induced HR did not. Our results suggest that a primary virulence target of these type‐III effectors resides in chloroplasts, and that the recognition of AvrRps4 by the plant immune system occurs elsewhere. Moreover, our results reveal that distinct type‐III effectors use a cleavable transit peptide to localize to chloroplasts, and that targets within this organelle are important for immunity.     

SPINDLY在壳寡糖诱导拟南芥抗丁香假单胞菌中的功能

为了探讨拟南芥O-岩藻糖基转移酶(SPINDLY)在病原体相关分子模式诱导抗性中的作用,该研究以SPINDLY缺失拟南芥突变体spy-3为实验材料,从叶片表型、病情指数、病菌定殖量以及丁香假单胞菌(Pst DC3000)关键基因的表达水平等指标,系统考察了SPINDLY在壳寡糖诱导拟南芥抗Pst DC3000中的功能。结果显示:(1)spy-3突变体比野生型更易被Pst DC3000侵染。(2)与病菌侵染组相比,壳寡糖预处理明显缓解植株叶片黄化现象,显著降低Pst DC3000的定殖量。(3)壳寡糖预处理的spy-3植株中水杨酸和茉莉酸途径相关基因的表达量及水杨酸和茉莉酸含量均较病菌侵染组明显升高。(4)壳寡糖在spy-3中的诱抗效果与野生型相比无明显差别。研究表明,SPINDLY在植物先天免疫过程发挥重要作用,但并不影响壳寡糖的诱导抗性。     

Tomato SlSAP3, a member of the stress-associated protein family,is a positive regulator of immunity against Pseudomonas syringae pv. tomato DC3000

Tomato stress-associated proteins (SAPs) belong to A20/AN1 zinc finger protein family, some of which have been shown to play important roles in plant stress responses. However, little is known about the functions and underlying molecular mechanisms of SAPs in plant immune responses. In the present study, we reported the function of tomato SlSAP3 in immunity to Pseudomonas syringae pv. tomato (Pst) DC3000. Silencing of SlSAP3 attenuated while overexpression of SlSAP3 in transgenic tomato increased immunity to Pst DC3000, accompanied with reduced and increased Pst DC3000-induced expression of SA signalling and defence genes, respectively. Flg22-induced reactive oxygen species (ROS) burst and expression of PAMP-triggered immunity (PTI) marker genes SlPTI5 and SlLRR22 were strengthened in SlSAP3-OE plants but were weakened in SlSAP3-silenced plants. SlSAP3 interacted with two SlBOBs and the A20 domain in SlSAP3 is critical for the SlSAP3-SlBOB1 interaction. Silencing of SlBOB1 and co-silencing of all three SlBOB genes conferred increased resistance to Pst DC3000, accompanied with increased Pst DC3000-induced expression of SA signalling and defence genes. These data demonstrate that SlSAP3 acts as a positive regulator of immunity against Pst DC3000 in tomato through the SA signalling and that SlSAP3 may exert its function in immunity by interacting with other proteins such as SlBOBs, which act as negative regulators of immunity against Pst DC3000 in tomato.     

Differential metabolomic responses of PAMP-triggered immunity and effector-triggered immunity in Arabidopsis suspension cells

Introduction

The rhizobacterial tomato pathogen Pseudomonas syringae pv. tomato str. DC3000 (PstDC3000), like many plant pathogenic bacteria, can elicit hypersensitive response in non-host plant cells. PstDC3000 uses a type III protein secretion system (T3SS) to deliver effector proteins.

Objectives

We compared metabolomic responses of Arabidopsis suspension cells to a wild-type PstDC3000, a T3SS deletion mutant PstDC3000D28E, and a pathogen associated molecular pattern (PAMP) flagellin’s N-terminal domain’s 22-aa peptide (flg22) to obtain metabolomics insights into the plant cell PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI).

Methods

Using targeted HPLC-MRM-MS and untargeted GC-MS approaches, we monitored qualitative and quantitative changes of 312 metabolites in central and specialized metabolic pathways in a time-course study.

Results

The overall metabolomic changes induced by the three treatments included phenylpropanoid, flavonoid, and phytohormone biosynthetic pathways, as well as primary metabolism in amino acid and sugar biosynthesis. In addition to shared metabolites, flg22, PstDC3000D28E and PstDC3000 each caused unique metabolite changes in the course of the development of PTI and ETI.

Conclusion

PstDC3000D28E triggered PTI responses were different from those of flg22. This study has not only revealed the discernible metabolomics features associated with the flg22, PstDC3000D28E and PstDC3000 treatments, but also laid a foundation toward further understanding of metabolic regulation and responses underlying plant PTI and ETI.

     

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.     

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.     

Functions of pipecolic acid on induced resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 in tomato plants

Amino acid metabolic pathways are involved in the plant immune system. Pipecolic acid (Pip), a lysine-derived non-protein amino acid, acts as an important regulator of disease resistance. Here, we report the functions of Pip on tomato disease resistance. Tomato seedlings treated with 0.5 mM Pip showed increased resistance to Pst DC3000 and B. cinerea compared with the control. After pathogen infection, the expression of defence-related genes increased in plants pretreated with Pip, while reactive oxygen species (ROS) accumulation decreased. These data demonstrated that exogenous application of Pip induced resistance against Pst DC3000 and B. cinerea in tomatoes, possibly through the regulation of ROS accumulation and defence-related gene expression.     

Rhizobacteria Bacillus subtilis restricts foliar pathogen entry through stomata

Plants exist in a complex multitrophic environment, where they interact with and compete for resources with other plants, microbes and animals. Plants have a complex array of defense mechanisms, such as the cell wall being covered with a waxy cuticle serving as a potent physical barrier. Although some pathogenic fungi infect plants by penetrating through the cell wall, many bacterial pathogens invade plants primarily through stomata on the leaf surface. Entry of the foliar pathogen, Pseudomonas syringae pathovar tomato DC3000 (hereafter PstDC3000), into the plant corpus occurs through stomatal openings, and consequently a key plant innate immune response is the transient closure of stomata, which delays disease progression. Here, we present evidence that the root colonization of the rhizobacteria Bacillus subtilis FB17 (hereafter FB17) restricts the stomata‐mediated pathogen entry of PstDC3000 in Arabidopsis thaliana. Root binding of FB17 invokes abscisic acid (ABA) and salicylic acid (SA) signaling pathways to close light‐adapted stomata. These results emphasize the importance of rhizospheric processes and environmental conditions as an integral part of the plant innate immune system against foliar bacterial infections.     

Synaptotagmin 5 Controls SYP132-VAMP721/722 Interaction for Arabidopsis Immunity to Pseudomonas syringae pv tomato DC3000

Vesicle-associated membrane proteins 721 and 722 (VAMP721/722) are secretory vesicle-localized arginine-conserved soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) to drive exocytosis in plants. They are involved in diverse physiological processes in plants by interacting with distinct plasma membrane (PM) syntaxins. Here, we show that synaptotagmin 5 (SYT5) is involved in plant defense against Pseudomonas syringae pv tomato (Pst) DC3000 by regulating SYP132-VAMP721/722 interactions. Calcium-dependent stimulation of in vitro SYP132-VAMP722 interaction by SYT5 and reduced in vivo SYP132-VAMP721/722 interaction in syt5 plants suggest that SYT5 regulates the interaction between SYP132 and VAMP721/722. We interestingly found that disease resistance to Pst DC3000 bacterium but not to Erysiphe pisi fungus is compromised in syt5 plants. Since SYP132 plays an immune function to bacteria, elevated growth of surface-inoculated Pst DC3000 in VAMP721/722-deficient plants suggests that SYT5 contributes to plant immunity to Pst DC3000 by promoting the SYP132-VAMP721/722 immune secretory pathway.     

High levels of cyclic‐di‐GMP in plant‐associated Pseudomonas correlate with evasion of plant immunity

The plant innate immune system employs plasma membrane‐localized receptors that specifically perceive pathogen/microbe‐associated molecular patterns (PAMPs/MAMPs). This induces a defence response called pattern‐triggered immunity (PTI) to fend off pathogen attack. Commensal bacteria are also exposed to potential immune recognition and must employ strategies to evade and/or suppress PTI to successfully colonize the plant. During plant infection, the flagellum has an ambiguous role, acting as both a virulence factor and also as a potent immunogen as a result of the recognition of its main building block, flagellin, by the plant pattern recognition receptors (PRRs), including FLAGELLIN SENSING2 (FLS2). Therefore, strict control of flagella synthesis is especially important for plant‐associated bacteria. Here, we show that cyclic‐di‐GMP [bis‐(3′‐5′)‐cyclic di‐guanosine monophosphate], a central regulator of bacterial lifestyle, is involved in the evasion of PTI. Elevated cyclic‐di‐GMP levels in the pathogen Pseudomonas syringae pv. tomato (Pto) DC3000, the opportunist P. aeruginosa PAO1 and the commensal P. protegens Pf‐5 inhibit flagellin synthesis and help the bacteria to evade FLS2‐mediated signalling in Nicotiana benthamiana and Arabidopsis thaliana. Despite this, high cellular cyclic‐di‐GMP concentrations were shown to drastically reduce the virulence of Pto DC3000 during plant infection. We propose that this is a result of reduced flagellar motility and/or additional pleiotropic effects of cyclic‐di‐GMP signalling on bacterial behaviour.     

Type one protein phosphatases (TOPPs) contribute to the plant defense response in Arabidopsis

Plant immunity must be tightly controlled to avoid activation of defense mechanisms in the absence of pathogen attack. Protein phosphorylation is a common mechanism regulating immune signaling. In Arabidopsis thaliana, nine members of the type one protein phosphatase (TOPP) family (also known as protein phosphatase 1, PP1) have been identified. Here, we characterized the autoimmune phenotype of topp4‐1, a previously identified dominant‐negative mutant of TOPP4. Epistasis analysis showed that defense activation in topp4‐1 depended on NON‐RACE‐SPECIFIC DISEASE RESISTANCE1, PHYTOALEXIN DEFICIENT4, and the salicylic acid pathway. We generated topp1/4/5/6/7/8/9 septuple mutants to investigate the function of TOPPs in plant immunity. Elevated defense gene expression and enhanced resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 in the septuple mutant indicate that TOPPs function in plant defense responses. Furthermore, TOPPs physically interacted with mitogen‐activated protein kinases (MAPKs) and affected the MAPK‐mediated downstream defense pathway. Thus, our study reveals that TOPPs are important regulators of plant immunity.     

Anti- and pro-microbial roles of autophagy in plant-bacteria interactions

Macroautophagy/autophagy and the ubiquitin-proteasome system (UPS) are major proteolytic pathways that are increasingly recognized as battlegrounds during host-microbe interactions in eukaryotes. In plants, the UPS has emerged as central component of innate immunity and is manipulated by bacterial pathogens to enhance virulence. Autophagy has been ascribed a similar importance for anti-bacterial immunity in animals, but the contribution of autophagy to host-bacteria interactions remained elusive in plants. Here, we present and discuss our recent findings that revealed anti- and pro-bacterial roles of autophagy pathways during bacterial infection in the model plant Arabidopsis thaliana. We discovered that selective autophagy mediated by the autophagy cargo receptor AT4G24690/NBR1 limits growth of Pseudomonas syringae pv. tomato DC3000 (Pst) by suppressing the establishment of an aqueous extracellular space (‘water-soaking’). In turn, Pseudomonas employs the effector protein HopM1 to activate autophagy and proteasome degradation (‘proteaphagy’), thereby enhancing its pathogenicity. Thus, our study demonstrates that distinct selective autophagy pathways contribute to host immunity and bacterial pathogenesis during Pst infection and provide evidence for an intimate crosstalk between the proteasome and autophagy system in plant-bacterial interactions.     

Role of ethylene signalling in growth and systemic resistance induction by the plant growth‐promoting fungus Penicillium viridicatum in Arabidopsis

The plant growth‐promoting fungi (PGPF) have long been known to improve plant growth and suppress plant diseases. The PGPF Penicillium viridicatum GP15‐1 elicited plant growth and induced systemic resistance (ISR) in Arabidopsis thaliana against Pseudomonas syringae pv. tomato DC3000 (Pst), leading to a restriction of pathogen growth and disease development. Examination of local and systemic genes indicated that GP15‐1 did not modulate the expression of any of the tested defence‐related marker genes involved in salicylic acid (SA), jasmonic acid (JA) and ethylene signalling pathways. Subsequent challenge of GP15‐1‐colonized plants with Pst bacterium primed Arabidopsis plants for enhanced activation of the JA‐inducible Atvsp (vegetative storage protein) gene at a later stage of infection. To assess the contribution of different signalling pathways in GP15‐1‐elicited plant growth and ISR, Arabidopsis genotypes implicated in SA signalling expressing the nahG transgene (NahG) or carrying disruption in NPR1 (npr1), JA signalling (jar1) and ethylene signalling (ein2) were tested. The GP15‐1‐induced plant growth and ISR were fully compromised in an ein2 mutation. Root colonization assay revealed that the inability of the ein2 mutant to express GP15‐1‐induced plant growth and ISR was not associated with reduced root colonization by GP15‐1. In conclusion, our results demonstrate the ethylene signalling pathway is involved in plant growth promotion and ISR elicitation by the PGPF P. viridicatum GP15‐1 in Arabidopsis. These results provide evidence that ethylene signalling has a substantial role in plant growth and disease resistance.