CBP60g and SARD1 play partially redundant critical roles in salicylic acid signaling

Arabidopsis thaliana calmodulin binding protein 60g (CBP60g) contributes to production of salicylic acid (SA) in response to recognition of microbe‐associated molecular patterns (MAMPs) such as flg22, a fragment of bacterial flagellin. Calmodulin binding is required for the function of CBP60g in limiting growth of the bacterial pathogen Pseudomonas syringae pv. maculicola (Pma) ES4326 and activation of SA synthesis. Here, we describe a closely related protein, SARD1. Unlike CBP60g, SARD1 does not bind calmodulin. Growth of Pma ES4326 is enhanced in sard1 mutants. In cbp60g sard1 double mutants, growth of Pma ES4326 is greatly enhanced, and SA levels and expression of PR‐1 and SID2 are dramatically reduced. Expression profiling placed the CBP60g/SARD1 node between the PAD4/EDS1 and SA nodes in the defense signaling network, and indicated that CBP60g and SARD1 affect defense responses in addition to SA production. A DNA motif bound by CBP60g and SARD1, GAAATTT, was significantly over‐represented in promoters of CBP60g/SARD1‐dependent genes, suggesting that expression of these genes is modulated by CBP60g/SARD1 binding. Gene expression patterns showed a stronger effect of cbp60g mutations soon after activation of a defense response, and a stronger effect of sard1 mutations at later times. The results are consistent with a model in which CBP60g and SARD1 comprise a partially redundant protein pair that is required for activation of SA production as well as other defense responses, with CBP60g playing a more important role early during the defense response, and SARD1 to playing a more important role later.     

Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae

Salicylic acid (SA)-induced defense responses are important factors during effector triggered immunity and microbe-associated molecular pattern (MAMP)-induced immunity in plants. This article presents evidence that a member of the Arabidopsis CBP60 gene family, CBP60g, contributes to MAMP-triggered SA accumulation. CBP60g is inducible by both pathogen and MAMP treatments. Pseudomonas syringae growth is enhanced in cbp60g mutants. Expression profiles of a cbp60g mutant after MAMP treatment are similar to those of sid2 and pad4, suggesting a defect in SA signaling. Accordingly, cbp60g mutants accumulate less SA when treated with the MAMP flg22 or a P. syringae hrcC strain that activates MAMP signaling. MAMP-induced production of reactive oxygen species and callose deposition are unaffected in cbp60g mutants. CBP60g is a calmodulin-binding protein with a calmodulin-binding domain located near the N-terminus. Calmodulin binding is dependent on Ca²⁺. Mutations in CBP60g that abolish calmodulin binding prevent complementation of the SA production and bacterial growth defects of cbp60g mutants, indicating that calmodulin binding is essential for the function of CBP60g in defense signaling. These studies show that CBP60g constitutes a Ca²⁺ link between MAMP recognition and SA accumulation that is important for resistance to P. syringae.     

Role of pathogen's effectors in understanding host-pathogen interaction

Pathogens can pose challenges to plant growth and development at various stages of their life cycle. Two interconnected defense strategies prevent the growth of pathogens in plants, i.e., molecular patterns triggered immunity (PTI) and pathogenic effector-triggered immunity (ETI) that often provides resistance when PTI no longer functions as a result of pathogenic effectors. Plants may trigger an ETI defense response by directly or indirectly detecting pathogen effectors via their resistance proteins. A typical resistance protein is a nucleotide-binding receptor with leucine-rich sequences (NLRs) that undergo structural changes as they recognize their effectors and form associations with other NLRs. As a result of dimerization or oligomerization, downstream components activate “helper” NLRs, resulting in a response to ETI. It was thought that ETI is highly dependent on PTI. However, recent studies have found that ETI and PTI have symbiotic crosstalk, and both work together to create a robust system of plant defense. In this article, we have summarized the recent advances in understanding the plant's early immune response, its components, and how they cooperate in innate defense mechanisms. Moreover, we have provided the current perspective on engineering strategies for crop protection based on up-to-date knowledge.     

Dynamics of Defense Responses and Cell Fate Change during Arabidopsis-Pseudomonas syringae Interactions

Plant-pathogen interactions involve sophisticated action and counteraction strategies from both parties. Plants can recognize pathogen derived molecules, such as conserved pathogen associated molecular patterns (PAMPs) and effector proteins, and subsequently activate PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI), respectively. However, pathogens can evade such recognitions and suppress host immunity with effectors, causing effector-triggered susceptibility (ETS). The differences among PTI, ETS, and ETI have not been completely understood. Toward a better understanding of PTI, ETS, and ETI, we systematically examined various defense-related phenotypes of Arabidopsis infected with different Pseudomonas syringae pv. maculicola ES4326 strains, using the virulence strain DG3 to induce ETS, the avirulence strain DG34 that expresses avrRpm1 (recognized by the resistance protein RPM1) to induce ETI, and HrcC^- that lacks the type three secretion system to activate PTI. We found that plants infected with different strains displayed dynamic differences in the accumulation of the defense signaling molecule salicylic acid, expression of the defense marker gene PR1, cell death formation, and accumulation/localization of the reactive oxygen species, H₂O₂. The differences between PTI, ETS, and ETI are dependent on the doses of the strains used. These data support the quantitative nature of PTI, ETS, and ETI and they also reveal qualitative differences between PTI, ETS, and ETI. Interestingly, we observed the induction of large cells in the infected leaves, most obviously with HrcC^- at later infection stages. The enlarged cells have increased DNA content, suggesting a possible activation of endoreplication. Consistent with strong induction of abnormal cell growth by HrcC^-, we found that the PTI elicitor flg22 also activates abnormal cell growth, depending on a functional flg22-receptor FLS2. Thus, our study has revealed a comprehensive picture of dynamic changes of defense phenotypes and cell fate determination during Arabidopsis-P. syringae interactions, contributing to a better understanding of plant defense mechanisms.     

Rpi-blb2-mediated late blight resistance in Nicotiana benthamiana requires SGT1 and salicylic acid-mediated signaling but not RAR1 or HSP90

The Rpi-blb2 recognizes the presence of the Phytophthora infestans AVRblb2 and initiates effector-triggered immunity (ETI). We performed gain-of-function and loss-of-function studies in Nicotiana benthamiana to elucidate Rpi-blb2-mediated resistance to P. infestans. Rpi-blb2 triggered a hypersensitive response through SGT1-mediated, but not RAR-mediated or HSP90-mediated, pathways. NbSGT1 was also required for basal and ETI-mediated by Rpi-blb2 in N. benthamiana. Moreover, salicylic acid (SA) affected basal defense and Rpi-blb2-mediated resistance against P. infestans. The increased susceptibility of Rpi-blb2-transgenic plants in the NahG-background correlated with reduced levels of SA. These findings provide evidence for the roles of SGT1- and SA-signaling in Rpi-blb2-mediated resistance against P. infestans.     

Plant autoimmunity—fresh insights into an old phenomenon

The plant immune system is well equipped to ward off the attacks of different types of phytopathogens. It primarily relies on two types of immune sensors—plasma membrane-resident receptor-like kinases and intracellular nucleotide-binding domain leucine-rich repeat (NLRs) receptors that engage preferentially in pattern- and effector-triggered immunity, respectively. Delicate fine-tuning, in particular of the NLR-governed branch of immunity, is key to prevent inappropriate and deleterious activation of plant immune responses. Inadequate NLR allele constellations, such as in the case of hybrid incompatibility, and the mis-activation of NLRs or the absence or modification of proteins guarded by these NLRs can result in the spontaneous initiation of plant defense responses and cell death—a phenomenon referred to as plant autoimmunity. Here, we review recent insights augmenting our mechanistic comprehension of plant autoimmunity. The recent findings broaden our understanding regarding hybrid incompatibility, unravel candidates for proteins likely guarded by NLRs and underline the necessity for the fine-tuning of NLR expression at various levels to avoid autoimmunity. We further present recently emerged tools to study plant autoimmunity and draw a cross-kingdom comparison to the role of NLRs in animal autoimmune conditions.

Increasing evidence indicates that plant autoimmunity is in many cases the consequence of the inappropriate activation of cytoplasmic immune sensors.     

BDNF-TrkB Axis Regulates Migration of the Lateral Line Primordium and Modulates the Maintenance of Mechanoreceptor Progenitors

BDNF and its specialized receptor TrkB are expressed in the developing lateral line system of zebrafish, but their role in this organ is unknown. To tackle this problem in vivo, we used transgenic animals expressing fluorescent markers in different cell types of the lateral line and combined a BDNF gain-of-function approach by BDNF mRNA overexpression and by soaking embryos in a solution of BDNF, with a loss-of-function approach by injecting the antisence ntrk2b-morpholino and treating embryos with the specific Trk inhibitor K252a. Subsequent analysis demonstrated that the BDNF-TrkB axis regulates migration of the lateral line primordium. In particular, BDNF-TrkB influences the expression level of components of chemokine signaling including Cxcr4b, and the generation of progenitors of mechanoreceptors, at the level of expression of Atoh1a-Atp2b1a.     

Different epitopes of Ralstonia solanacearum effector RipAW are recognized by two Nicotiana species and trigger immune responses

Diverse pathogen effectors convergently target conserved components in plant immunity guarded by intracellular nucleotide-binding domain leucine-rich repeat receptors (NLRs) and activate effector-triggered immunity (ETI), often causing cell death. Little is known of the differences underlying ETI in different plants triggered by the same effector. In this study, we demonstrated that effector RipAW triggers ETI on Nicotiana benthamiana and Nicotiana tabacum. Both the first 107 amino acids (N_1-107) and RipAW E3-ligase activity are required but not sufficient for triggering ETI on N. benthamiana. However, on N. tabacum, the N_1-107 fragment is essential and sufficient for inducing cell death. The first 60 amino acids of the protein are not essential for RipAW-triggered cell death on either N. benthamiana or N. tabacum. Furthermore, simultaneous mutation of both R75 and R78 disrupts RipAW-triggered ETI on N. tabacum, but not on N. benthamiana. In addition, N. tabacum recognizes more RipAW orthologs than N. benthamiana. These data showcase the commonalities and specificities of RipAW-activated ETI in two evolutionally related species, suggesting Nicotiana species have acquired different abilities to perceive RipAW and activate plant defences during plant–pathogen co-evolution.     

CPR5 positively regulates pattern-triggered immunity via a mediator protein

Plant cells possess a two-layered immune system consisting of pattern-triggered immunity(PTI)and effector-triggered immunity(ETI), mediated by cell surface pattern-recognition receptors and intracellular nucleotide-binding leucine-rich repeat receptors(NLRs), respectively. The CONSTITUTIVE EXPRESSION OF PR GENES 5(CPR5) nuclear pore complex protein negatively regulates ETI, including ETI-associated hypersensitive response. Here, we show that CPR5 is essential for the activation of various PTI re...     

Diverse Roles of the Salicylic Acid Receptors NPR1 and NPR3/NPR4 in Plant Immunity

The plant defense hormone salicylic acid (SA) is perceived by two classes of receptors, NPR1 and NPR3/NPR4. They function in two parallel pathways to regulate SA-induced defense gene expression. To better understand the roles of the SA receptors in plant defense, we systematically analyzed their contributions to different aspects of Arabidopsis (Arabidopsis thaliana) plant immunity using the SA-insensitive npr1-1 npr4-4D double mutant. We found that perception of SA by NPR1 and NPR4 is required for activation of N-hydroxypipecolic acid biosynthesis, which is essential for inducing systemic acquired resistance. In addition, both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) are severely compromised in the npr1-1 npr4-4D double mutant. Interestingly, the PTI and ETI attenuation in npr1-1 npr4-4D is more dramatic compared with the SA-induction deficient2-1 (sid2-1) mutant, suggesting that the perception of residual levels of SA in sid2-1 also contributes to immunity. Furthermore, NPR1 and NPR4 are involved in positive feedback amplification of SA biosynthesis and regulation of SA homeostasis through modifications including 5-hydroxylation and glycosylation. Thus, the SA receptors NPR1 and NPR4 play broad roles in plant immunity.     

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.     

Pto Kinase Binds Two Domains of AvrPtoB and Its Proximity to the Effector E3 Ligase Determines if It Evades Degradation and Activates Plant Immunity

The tomato—Pseudomonas syringae pv. tomato (Pst)—pathosystem is one of the best understood models for plant-pathogen interactions. Certain wild relatives of tomato express two closely related members of the same kinase family, Pto and Fen, which recognize the Pst virulence protein AvrPtoB and activate effector-triggered immunity (ETI). AvrPtoB, however, contains an E3 ubiquitin ligase domain in its carboxyl terminus which causes degradation of Fen and undermines its ability to activate ETI. In contrast, Pto evades AvrPtoB-mediated degradation and triggers ETI in response to the effector. It has been reported recently that Pto has higher kinase activity than Fen and that this difference allows Pto to inactivate the E3 ligase through phosphorylation of threonine-450 (T450) in AvrPtoB. Here we show that, in contrast to Fen which can only interact with a single domain proximal to the E3 ligase of AvrPtoB, Pto binds two distinct domains of the effector, the same site as Fen and another N-terminal domain. In the absence of E3 ligase activity Pto binds to either domain of AvrPtoB to activate ETI. However, the presence of an active E3 ligase domain causes ubiquitination of Pto that interacts with the domain proximal to the E3 ligase, identical to ubiquitination of Fen. Only when Pto binds its unique distal domain can it resist AvrPtoB-mediated degradation and activate ETI. We show that phosphorylation of T450 is not required for Pto-mediated resistance in vivo and that a kinase-inactive version of Pto is still capable of activating ETI in response to AvrPtoB. Our results demonstrate that the ability of Pto to interact with a second site distal to the E3 ligase domain in AvrPtoB, and not a higher kinase activity or T450 phosphorylation, allows Pto to evade ubiquitination and to confer immunity to Pst.     

STAND alert! Prokaryotic immunity for recognition and defense against bacteriophages

In a recent article, Gao et al. diversify our knowledge of prokaryotic innate immunity by characterizing a novel bacterial defense system that utilizes nucleotide-binding oligomerization-like receptors (NLRs) for recognizing phage proteins.     

Role of Rip2 in Development of Tumor-Infiltrating MDSCs and Bladder Cancer Metastasis

Tumor invasion and metastases represent a complex series of molecular events that portends a poor prognosis. The contribution of inflammatory pathways mediating this process is not well understood. Nod-like receptors (NLRs) of innate immunity function as intracellular sensors of pathogen motifs and danger molecules. We propose a role of NLRs in tumor surveillance and in programming tumor-infiltrating lymphocytes (TILs). In this study, we examined the downstream serine/threonine and tyrosine kinase Rip2 in a murine model of bladder cancer. In Rip2-deficient C57Bl6 mice, larger orthotopic MB49 tumors developed with more numerous and higher incidence of metastases compared to wild-type controls. As such, increased tumor infiltration of CD11b⁺Gr1^hi myeloid-derived suppressor cells (MDSCs) with concomitant decrease in T cells and NK cells were observed in Rip2-deficient tumor bearing animals using orthotopic and subcutaneous tumor models. Rip2-deficient tumors showed enhanced epithelial-to-mesenchymal transition, with elevated expression of zeb1, zeb2, twist, and snail in the tumor microenvironment. We found that the absence of Rip2 plays an intrinsic role in fostering the development of granulocytic MDSCs by an autocrine and paracrine effect of granulocytic colony stimulating factor (G-CSF) expression. Our findings suggest that NLR pathways may be a novel modality to program TILs and influence tumor metastases.