Colour-analyzer: a new dual colour model-based imaging tool to quantify plant disease.

BACKGROUND: Despite major efforts over the last decades, the rising demands of the growing global population makes it of paramount importance to increase crop yields and reduce losses caused by plant pathogens. One way to tackle this is to screen novel resistant genotypes and immunity-inducing agents, which must be conducted in a high-throughput manner. RESULTS: Colour-analyzer is a free web-based tool that can be used to rapidly measure the formation of lesions on leaves. Pixel colour values are often used to distinguish infected from healthy tissues. Some programs employ colour models, such as RGB, HSV or L*a*b*. Colour-analyzer uses two colour models, utilizing both HSV (Hue, Saturation, Value) and L*a*b* values. We found that the a* b* values of the L*a*b* colour model provided the clearest distinction between infected and healthy tissue, while the H and S channels were best to distinguish the leaf area from the background. CONCLUSION: By combining the a* and b* channels to determine the lesion area, while using the H and S channels to determine the leaf area, Colour-analyzer provides highly accurate information on the size of the lesion as well as the percentage of infected tissue in a high throughput manner and can accelerate the plant immunity research field.

A tale of many families: calcium channels in plant immunity.

Plants launch a concerted immune response to dampen potential infections upon sensing microbial pathogen and insect invasions. The transient and rapid elevation of the cytosolic calcium concentration [Ca2+]cyt is among the essential early cellular responses in plant immunity. The free Ca2+ concentration in the apoplast is far higher than that in the resting cytoplasm. Thus, the precise regulation of calcium channel activities upon infection is the key for an immediate and dynamic Ca2+ influx to trigger downstream signaling. Specific Ca2+ signatures in different branches of the plant immune system vary in timing, amplitude, duration, kinetics, and sources of Ca2+. Recent breakthroughs in the studies of diverse groups of classical calcium channels highlight the instrumental role of Ca2+ homeostasis in plant immunity and cell survival. Additionally, the identification of some immune receptors as noncanonical Ca2+-permeable channels opens a new view of how immune receptors initiate cell death and signaling. This review aims to provide an overview of different Ca2+-conducting channels in plant immunity and highlight their molecular and genetic mode-of-actions in facilitating immune signaling. We also discuss the regulatory mechanisms that control the stability and activity of these channels.

Arabidopsis thaliana CYCLIC NUCLEOTIDE-GATED CHANNEL2 mediates extracellular ATP signal transduction in root epidermis.

Damage can be signalled by extracellular ATP (eATP) using plasma membrane (PM) receptors to effect cytosolic free calcium ion ([Ca2+ ]cyt ) increase as a second messenger. The downstream PM Ca2+ channels remain enigmatic. Here, the Arabidopsis thaliana Ca2+ channel subunit CYCLIC NUCLEOTIDE-GATED CHANNEL2 (CNGC2) was identified as a critical component linking eATP receptors to downstream [Ca2+ ]cyt signalling in roots. Extracellular ATP-induced changes in single epidermal cell PM voltage and conductance were measured electrophysiologically, changes in root [Ca2+ ]cyt were measured with aequorin, and root transcriptional changes were determined by quantitative real-time PCR. Two cngc2 loss-of-function mutants were used: cngc2-3 and defence not death1 (which expresses cytosolic aequorin). Extracellular ATP-induced transient depolarization of Arabidopsis root elongation zone epidermal PM voltage was Ca2+ dependent, requiring CNGC2 but not CNGC4 (its channel co-subunit in immunity signalling). Activation of PM Ca2+ influx currents also required CNGC2. The eATP-induced [Ca2+ ]cyt increase and transcriptional response in cngc2 roots were significantly impaired. CYCLIC NUCLEOTIDE-GATED CHANNEL2 is required for eATP-induced epidermal Ca2+ influx, causing depolarization leading to [Ca2+ ]cyt increase and damage-related transcriptional response.

Auxin analog-induced Ca2+ signaling is independent of inhibition of endosomal aggregation in Arabidopsis roots.

Much of what we know about the role of auxin in plant development derives from exogenous manipulations of auxin distribution and signaling, using inhibitors, auxins, and auxin analogs. In this context, synthetic auxin analogs, such as 1-naphthalene acetic acid (1-NAA), are often favored over the endogenous auxin, indole-3-acetic acid (IAA), in part due to their higher stability. While such auxin analogs have proven instrumental in revealing the various faces of auxin, they display in some cases bioactivities distinct from IAA. Here, we focused on the effect of auxin analogs on the accumulation of PIN proteins in brefeldin A-sensitive endosomal aggregations (BFA bodies), and correlation with the ability to elicit Ca2+ responses. For a set of commonly used auxin analogs, we evaluated if auxin analog-induced Ca2+ signaling inhibits PIN accumulation. Not all auxin analogs elicited a Ca2+ response, and their differential ability to elicit Ca2+ responses correlated partially with their ability to inhibit BFA-body formation. However, in tir1/afb and cngc14, 1-NAA-induced Ca2+ signaling was strongly impaired, yet 1-NAA still could inhibit PIN accumulation in BFA bodies. This demonstrates that TIR1/AFB-CNGC14-dependent Ca2+ signaling does not inhibit BFA body formation in Arabidopsis roots.

CYCLIC NUCLEOTIDE-GATED ION CHANNEL 2 modulates auxin homeostasis and signaling.

Cyclic nucleotide-gated ion channels (CNGCs) have been firmly established as Ca2+-conducting ion channels that regulate a wide variety of physiological responses in plants. CNGC2 has been implicated in plant immunity and Ca2+ signaling due to the autoimmune phenotypes exhibited by null mutants of CNGC2 in Arabidopsis thaliana. However, cngc2 mutants display additional phenotypes that are unique among autoimmune mutants, suggesting that CNGC2 has functions beyond defense and generates distinct Ca2+ signals in response to different triggers. In this study, we found that cngc2 mutants showed reduced gravitropism, consistent with a defect in auxin signaling. This was mirrored in the diminished auxin response detected by the auxin reporters DR5::GUS and DII-VENUS and in a strongly impaired auxin-induced Ca2+ response. Moreover, the cngc2 mutant exhibits higher levels of the endogenous auxin indole-3-acetic acid, indicating that excess auxin in the cngc2 mutant causes its pleiotropic phenotypes. These auxin signaling defects and the autoimmunity syndrome of the cngc2 mutant could be suppressed by loss-of-function mutations in the auxin biosynthesis gene YUCCA6 (YUC6), as determined by identification of the cngc2 suppressor mutant repressor of cngc2 (rdd1) as an allele of YUC6. A loss-of-function mutation in the upstream auxin biosynthesis gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA1, WEAK ETHYLENE INSENSITIVE8) also suppressed the cngc2 phenotypes, further supporting the tight relationship between CNGC2 and the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS-YUCCA -dependent auxin biosynthesis pathway. Taking these results together, we propose that the Ca2+ signal generated by CNGC2 is a part of the negative feedback regulation of auxin homeostasis in which CNGC2 balances cellular auxin perception by influencing auxin biosynthesis.

Impact of Maternal Intrapartum Antibiotics, and Caesarean Section with and without Labour on Bifidobacterium and Other Infant Gut Microbiota.

BACKGROUND AND AIMS: Few studies consider the joint effect of multiple factors related to birth, delivery mode, intrapartum antibiotic prophylaxis and the onset of labour, on the abundance of Bifidobacterium and the quantity of this genus and its species Bifidobacterium longum subsp. infantis in the infant gut microbiota. We implemented such a study. METHODS: Among 1654 Canadian full-term infants, the gut microbiota of faecal samples collected at 3 months were profiled by 16S rRNA sequencing; the genus Bifidobacterium and Bifidobacterium longum subsp. infantis were quantified by qPCR. Associations between Bifidobacterium and other gut microbiota were examined by Spearman's rank correlation. RESULTS: Following vaginal birth, maternal IAP exposure was associated with reduced absolute quantities of bifidobacteria among vaginally delivered infants (6.80 vs. 7.14 log10 (gene-copies/g faeces), p < 0.05), as well as their lowered abundance relative to other gut microbiota. IAP differences in infant gut bifidobacterial quantity were independent of maternal pre-pregnancy body-mass-index (BMI), and remarkably, they were limited to breastfed infants. Pre-pregnancy BMI adjustment revealed negative associations between absolute quantities of bifidobacteria and CS with or without labour in non-breastfed infants, and CS with labour in exclusively breastfed infants. Significant correlations between Bifidobacterium abundance and other microbial taxa were observed. CONCLUSIONS: This study documented the impact of the birth mode and feeding status on the abundance of gut Bifidobacterium, and pointed to the important ecological role of the genus Bifidobacterium in gut microbiota due to its strong interaction with other gut microbiota in early infancy.

Multiple phosphorylation events of the mitochondrial membrane protein TTM1 regulate cell death during senescence.

The role of mitochondria in programmed cell death (PCD) during animal growth and development is well documented, but much less is known for plants. We previously showed that the Arabidopsis thaliana triphosphate tunnel metalloenzyme (TTM) proteins TTM1 and TTM2 are tail-anchored proteins that localize in the mitochondrial outer membrane and participate in PCD during senescence and immunity, respectively. Here, we show that TTM1 is specifically involved in senescence induced by abscisic acid (ABA). Moreover, phosphorylation of TTM1 by multiple mitogen-activated protein (MAP) kinases regulates its function and turnover. A combination of proteomics and in vitro kinase assays revealed three major phosphorylation sites of TTM1 (Ser10, Ser437, and Ser490). Ser437, which is phosphorylated upon perception of senescence cues such as ABA and prolonged darkness, is phosphorylated by the MAP kinases MPK3 and MPK4, and Ser437 phosphorylation is essential for TTM1 function in senescence. These MPKs, together with three additional MAP kinases (MPK1, MPK7, and MPK6), also phosphorylate Ser10 and Ser490, marking TTM1 for protein turnover, which likely prevents uncontrolled cell death. Taken together, our results show that multiple MPKs regulate the function and turnover of the mitochondrial protein TTM1 during senescence-associated cell death, revealing a novel link between mitochondria and PCD.

The Receptor Kinases BAK1/SERK4 Regulate Ca2+ Channel-Mediated Cellular Homeostasis for Cell Death Containment.

Cell death is a vital and ubiquitous process that is tightly controlled in all organisms. However, the mechanisms underlying precise cell death control remain fragmented. As an important shared module in plant growth, development, and immunity, Arabidopsis thaliana BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1) and somatic embryogenesis receptor kinase 4 (SERK4) redundantly and negatively regulate plant cell death. By deploying an RNAi-based genetic screen for bak1/serk4 cell death suppressors, we revealed that cyclic nucleotide-gated channel 20 (CNGC20) functions as a hyperpolarization-activated Ca2+-permeable channel specifically regulating bak1/serk4 cell death. BAK1 directly interacts with and phosphorylates CNGC20 at specific sites in the C-terminal cytosolic domain, which in turn regulates CNGC20 stability. CNGC19, the closest homolog of CNGC20 with a low abundance compared with CNGC20, makes a quantitative genetic contribution to bak1/serk4 cell death only in the absence of CNGC20, supporting the biochemical data showing homo- and heteromeric assembly of the CNGC20 and CNGC19 channel complexes. Transcripts of CNGC20 and CNGC19 are elevated in bak1/serk4 compared with wild-type plants, further substantiating a critical role of homeostasis of CNGC20 and CNGC19 in cell death control. Our studies not only uncover a unique regulation of ion channel stability by cell-surface-resident receptor kinase-mediated phosphorylation but also provide evidence for fine-tuning Ca2+ channel functions in maintaining cellular homeostasis by the formation of homo- and heterotetrameric complexes.

A host-pathogen interactome uncovers phytopathogenic strategies to manipulate plant ABA responses.

The phytopathogen Pseudomonas syringae delivers into host cells type III secreted effectors (T3SEs) that promote virulence. One virulence mechanism employed by T3SEs is to target hormone signaling pathways to perturb hormone homeostasis. The phytohormone abscisic acid (ABA) influences interactions between various phytopathogens and their plant hosts, and has been shown to be a target of P. syringae T3SEs. In order to provide insight into how T3SEs manipulate ABA responses, we generated an ABA-T3SE interactome network (ATIN) between P. syringae T3SEs and Arabidopsis proteins encoded by ABA-regulated genes. ATIN consists of 476 yeast-two-hybrid interactions between 97 Arabidopsis ABA-regulated proteins and 56 T3SEs from four pathovars of P. syringae. We demonstrate that T3SE interacting proteins are significantly enriched for proteins associated with transcription. In particular, the ETHYLENE RESPONSIVE FACTOR (ERF) family of transcription factors is highly represented. We show that ERF105 and ERF8 displayed a role in defense against P. syringae, supporting our overall observation that T3SEs of ATIN converge on proteins that influence plant immunity. In addition, we demonstrate that T3SEs that interact with a large number of ABA-regulated proteins can influence ABA responses. One of these T3SEs, HopF3Pph6 , inhibits the function of ERF8, which influences both ABA-responses and plant immunity. These results provide a potential mechanism for how HopF3Pph6 manipulates ABA-responses to promote P. syringae virulence, and also demonstrate the utility of ATIN as a resource to study the ABA-T3SE interface.

Ca2+ to the rescue - Ca2+channels and signaling in plant immunity.

Ca2+ is a universal second messenger in many signaling pathways in all eukaryotes including plants. Transient changes in [Ca2+]cyt are rapidly generated upon a diverse range of stimuli such as drought, heat, wounding, and biotic stresses (infection by pathogenic and symbiotic microorganisms), as well as developmental cues. It has been known for a while that [Ca2+]cyt transient signals play crucial roles to activate plant immunity and recently significant progresses have been made in this research field. However the identity and regulation of ion channels that are involved in defense related Ca2+ signals are still enigmatic. Members of two ligand gated ion channel families, glutamate receptor-like channels (GLRs) and cyclic nucleotide-gated channels (CNGCs) have been implicated in immune responses; nevertheless more precise data to understand their direct involvement in the creation of Ca2+ signals during immune responses is necessary. Furthermore, the study of other ion channel groups is also required to understand the whole picture of the intra- and inter-cellular Ca2+ signalling network. In this review we summarize Ca2+ signals in plant immunity from an ion channel point of view and discuss future challenges in this exciting research field.

Arabidopsis ETHYLENE RESPONSE FACTOR 8 (ERF8) has dual functions in ABA signaling and immunity.

BACKGROUND: ETHYLENE RESPONSE FACTOR (ERF) 8 is a member of one of the largest transcription factor families in plants, the APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) superfamily. Members of this superfamily have been implicated in a wide variety of processes such as development and environmental stress responses. RESULTS: In this study we demonstrated that ERF8 is involved in both ABA and immune signaling. ERF8 overexpression induced programmed cell death (PCD) in Arabidopsis and Nicotiana benthamiana. This PCD was salicylic acid (SA)-independent, suggesting that ERF8 acts downstream or independent of SA. ERF8-induced PCD was abolished by mutations within the ERF-associated amphiphilic repression (EAR) motif, indicating ERF8 induces cell death through its transcriptional repression activity. Two immunity-related mitogen-activated protein kinases, MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) and MPK11, were identified as ERF8-interacting proteins and directly phosphorylated ERF8 in vitro. Four putative MPK phosphorylation sites were identified in ERF8, one of which (Ser103) was determined to be the predominantly phosphorylated residue in vitro, while mutation of all four putative phosphorylation sites partially suppressed ERF8-induced cell death in N. benthamiana. Genome-wide transcriptomic analysis and pathogen growth assays confirmed a positive role of ERF8 in mediating immunity, as ERF8 knockdown or overexpression lines conferred compromised or enhanced resistance against the hemibiotrophic bacterial pathogen Pseudomonas syringae, respectively. CONCLUSIONS: Together these data reveal that the ABA-inducible transcriptional repressor ERF8 has dual roles in ABA signaling and pathogen defense, and further highlight the complex influence of ABA on plant-microbe interactions.

Triphosphate Tunnel Metalloenzyme Function in Senescence Highlights a Biological Diversification of This Protein Superfamily.

The triphosphate tunnel metalloenzyme (TTM) superfamily comprises a group of enzymes that hydrolyze organophosphate substrates. They exist in all domains of life, yet the biological role of most family members is unclear. Arabidopsis (Arabidopsis thaliana) encodes three TTM genes. We have previously reported that AtTTM2 displays pyrophosphatase activity and is involved in pathogen resistance. Here, we report the biochemical activity and biological function of AtTTM1 and diversification of the biological roles between AtTTM1 and 2 Biochemical analyses revealed that AtTTM1 displays pyrophosphatase activity similar to AtTTM2, making them the only TTMs characterized so far to act on a diphosphate substrate. However, knockout mutant analysis showed that AtTTM1 is not involved in pathogen resistance but rather in leaf senescence. AtTTM1 is transcriptionally up-regulated during leaf senescence, and knockout mutants of AtTTM1 exhibit delayed dark-induced and natural senescence. The double mutant of AtTTM1 and AtTTM2 did not show synergistic effects, further indicating the diversification of their biological function. However, promoter swap analyses revealed that they functionally can complement each other, and confocal microscopy revealed that both proteins are tail-anchored proteins that localize to the mitochondrial outer membrane. Additionally, transient overexpression of either gene in Nicotiana benthamiana induced senescence-like cell death upon dark treatment. Taken together, we show that two TTMs display the same biochemical properties but distinct biological functions that are governed by their transcriptional regulation. Moreover, this work reveals a possible connection of immunity-related programmed cell death and senescence through novel mitochondrial tail-anchored proteins.

Using GCaMP3 to Study Ca2+ Signaling in Nicotiana Species.

Ca2+ signaling is a central component of plant biology; however, direct analysis of in vivo Ca2+ levels is experimentally challenging. In recent years, the use of genetically encoded Ca2+ indicators has revolutionized the study of plant Ca2+ signaling, although such studies have been largely restricted to the model plant Arabidopsis. We have developed stable transgenic Nicotiana benthamiana and Nicotiana tabacum lines expressing the single-wavelength fluorescent Ca2+ indicator, GCaMP3. Ca2+ levels in these plants can be imaged in situ using fluorescence microscopy, and these plants can be used qualitatively and semi-quantitatively to evaluate Ca2+ signals in response to a broad array of abiotic or biotic stimuli, such as cold shock or pathogen-associated molecular patterns (PAMPs). Furthermore, these tools can be used in conjunction with well-established N. benthamiana techniques such as virus-induced gene silencing (VIGS) or transient heterologous expression to assay the effects of loss or gain of function on Ca2+ signaling, an approach which we validated via silencing or transient expression of the PAMP receptors FLS2 (Flagellin Sensing 2) or EFR (EF-Tu receptor), respectively. Using these techniques, along with chemical inhibitor treatments, we demonstrate how these plants can be used to elucidate the molecular components governing Ca2+ signaling in response to specific stimuli.

Calmodulin as a Ca2+-Sensing Subunit of Arabidopsis Cyclic Nucleotide-Gated Channel Complexes.

Ca2+ serves as a universal second messenger in eukaryotic signaling pathways, and the spatial and temporal patterns of Ca2+ concentration changes are determined by feedback and feed-forward regulation of the involved transport proteins. Cyclic nucleotide-gated channels (CNGCs) are Ca2+-permeable channels that interact with the ubiquitous Ca2+ sensor calmodulin (CaM). CNGCs interact with CaMs via diverse CaM-binding sites, including an IQ-motif, which has been identified in the C-termini of CNGC20 and CNGC12. Here we present a family-wide analysis of the IQ-motif from all 20 Arabidopsis CNGC isoforms. While most of their IQ-peptides interacted with conserved CaMs in yeast, some were unable to do so, despite high sequence conservation across the family. We showed that the CaM binding ability of the IQ-motif is highly dependent on its proximal and distal vicinity. We determined that two alanine residues positioned N-terminal to the core IQ-sequence play a significant role in CaM binding, and identified a polymorphism at this site that promoted or inhibited CaM binding in yeast. Through detailed biophysical analysis of the CNGC2 IQ-motif, we found that this polymorphism specifically affected the Ca2+-independent interactions with the C-lobe of CaM. This same polymorphism partially suppressed the induction of programmed cell death by CNGC11/12 in planta. Our work expands the model of CNGC regulation, and posits that the C-lobe of apo-CaM is permanently associated with the channel at the N-terminal part of the IQ-domain. This mode allows CaM to function as a Ca2+-sensing regulatory subunit of the channel complex, providing a mechanism by which Ca2+ signals may be fine-tuned.

Opening the Gates: Insights into Cyclic Nucleotide-Gated Channel-Mediated Signaling.

Recent work has expanded our understanding of the roles of cyclic nucleotide-gated channels (CNGCs) in plant signaling. In this spotlight article, we discuss advances and future perspectives in determining how CNGCs mediate calcium signaling in response to diverse stimuli.

Multiple Calmodulin-Binding Sites Positively and Negatively Regulate Arabidopsis CYCLIC NUCLEOTIDE-GATED CHANNEL12.

Ca(2+) signaling is critical to plant immunity; however, the channels involved are poorly characterized. Cyclic nucleotide-gated channels (CNGCs) are nonspecific, Ca(2+)-permeable cation channels. Plant CNGCs are hypothesized to be negatively regulated by the Ca(2+) sensor calmodulin (CaM), and previous work has focused on a C-terminal CaM-binding domain (CaMBD) overlapping with the cyclic nucleotide binding domain of plant CNGCs. However, we show that the Arabidopsis thaliana isoform CNGC12 possesses multiple CaMBDs at cytosolic N and C termini, which is reminiscent of animal CNGCs and unlike any plant channel studied to date. Biophysical characterizations of these sites suggest that apoCaM interacts with a conserved isoleucine-glutamine (IQ) motif in the C terminus of the channel, while Ca(2+)/CaM binds additional N- and C-terminal motifs with different affinities. Expression of CNGC12 with a nonfunctional N-terminal CaMBD constitutively induced programmed cell death, providing in planta evidence of allosteric CNGC regulation by CaM. Furthermore, we determined that CaM binding to the IQ motif was required for channel function, indicating that CaM can both positively and negatively regulate CNGC12. These data indicate a complex mode of plant CNGC regulation by CaM, in contrast to the previously proposed competitive ligand model, and suggest exciting parallels between plant and animal channels.