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Key messageA point mutation of RPM1 triggers persistent immune response that induces leaf premature senescence in wheat, providing novel information of immune responses and leaf senescence.Leaf premature senescence in wheat (Triticum aestivum L.) is one of the most common factors affecting the plant's development and yield. In this study, we identified a novel wheat mutant, yellow leaf and premature senescence (ylp), which exhibits yellow leaves and premature senescence at the heading and flowering stages. Consistent with the yellow leaves phenotype, ylp had damaged and collapsed chloroplasts. Map-based cloning revealed that the phenotype of ylp was caused by a point mutation from Arg to His at amino acid 790 in a plasma membrane–localized protein resistance to Pseudomonas syringae pv. maculicola 1 (RPM1). The point mutation triggered excessive immune responses and the upregulation of senescence- and autophagy-associated genes. This work provided the information for understanding the molecular regulatory mechanism of leaf senescence, and the results would be important to analyze which mutations of RPM1 could enable plants to obtain immune activation without negative effects on plant growth.

In this paper we describe PATTERN-TRIGGERED IMMUNITY (PTI) COMPROMISED RECEPTOR-LIKE CYTOPLASMIC KINASE 1 (PCRK1) of Arabidopsis thaliana, an RLCK that is important for defense against the pathogen Pseudomonas syringae pv. maculicola ES4326 (Pma ES4326). We examined defense responses such as bacterial growth, production of reactive oxygen species (ROS) and callose deposition in pcrk1 mutant plants to determine the role of PCRK1 during pathogen infection. Expression of PCRK1 was induced following pathogen infection. Pathogen growth was significantly higher in pcrk1 mutant lines than in wild-type Col-0. Mutant pcrk1 plants showed reduced pattern-triggered immunity (PTI) against Pma ES4326 after pretreatment with peptides derived from flagellin (flg22), elongation factor-Tu (elf18), or an endogenous protein (pep1). Deposition of callose was reduced in pcrk1 plants, indicating a role of PCRK1 in activation of early immune responses. A PCRK1 transgene containing a mutation in a conserved lysine residue important for phosphorylation activity of kinases (K118E) failed to complement a pcrk1 mutant for the Pma ES4326 growth phenotype. Our study shows that PCRK1 plays an important role during PTI and that a conserved lysine residue in the putative kinase domain is important for PCRK1 function.

Invasive bacteria have caused tremendous losses to global ecosystems and agricultural production, yet effective control measures remain elusive. Plant specialized metabolites are being investigated as an important source of antimicrobial active substances. And Ailanthus altissima is an abundant tree widespread throughout Northeast China. In this study, we identified 21 compounds from A. altissima leaves, including steroids, terpenes, phenolics, and coumarins. Two new steroidal compounds, ailanstigol A (1) and ailanstigol B (2), and one new coumarin (2′R,3′R)-7-(2′,3′,6′-trihydroxy-3′-methylhexyloxy)-6,8-dimethoxycoumarin (3) were isolated. Antibacterial screening revealed that compounds 1 and 2 exhibited inhibitory activity against two invasive bacteria, Xanthomonas oryzae pv. oryzae PXO 71A and PXO 86A and Pseudomonas syringae pv. maculicola ES4326. Further mechanistic screening unveiled that the steroidal compounds 1 and 2 may inhibit bacterial growth and reproduction by reducing cell viability, disrupting the cell membrane and increasing protein leakage, and inhibiting biofilm formation. In summary, our results enriched the known chemical diversity of A. altissima and provided a foundation for investigating the mechanisms by which steroidal compounds inhibit invasive bacterial growth.

Recently, the octameric vesicle-tethering complex exocyst was found in plants and its importance for Arabidopsis morphogenesis was demonstrated. Exo70 exocyst subunits in plants, unlike in yeasts and mammals, are represented by a multigene family, comprising 23 members in Arabidopsis. For Exo70B2 and Exo70H1 paralogues, transcriptional up-regulation was confirmed on treatment with an elicitor peptide, elf18, derived from the bacterial elongation factor. Their ability to participate in the exocyst complex formation was inferred by the interaction of both the Exo70s with several other exocyst subunits using the yeast two-hybrid system. Arabidopsis plants mutated in these two genes were used to analyse their local reaction upon inoculation with Pseudomonas syringae pv. maculicola and the fungal pathogen Blumeria graminis f. sp. hordei. The Pseudomonas sensitivity test revealed enhanced susceptibility for the two exo70B2 and one H1 mutant lines. After Blumeria inoculation, an increase in the proportion of abnormal papilla formation, with an unusual wide halo made of vesicle-like structures, was found in exo70B2 mutants. Intracellular localization of both Exo70 proteins was studied following a GFP fusion assay and Agrobacterium-mediated transient expression of the constructs in Nicotiana benthamiana leaf epidermis. GFP-Exo70H1 localizes in the vesicle-like structures, while GFP-Exo70B2 is localized mainly in the cytoplasm. It is concluded that both Exo70B2 and Exo70H1 are involved in the response to pathogens, with Exo70B2 having a more important role in cell wall apposition formation related to plant defence.

Three semi-selective media, DTarTA, SPbc, and SPamt, were developed and tested to isolate Pseudomonas syringae pv. maculicola ( Psm ) and P . cannabina pv. alisalensis ( Pca ) from Raphanus sativus seeds. DTarTA contained D-tartaric acid as a carbon source and potassium tellurite, ampicillin sodium, and methyl violet as antibiotics. DTarTA suppressed growth in 19 of the 24 pathovars from the P . syringae complex, whereas Psm and Pca grew and formed gray to black colonies. SPamt contained sucrose and peptone as nutrient sources and was supplemented with bromothymol blue and the same antibiotics present in DTarTA and Psm and Pca formed yellowish to dark brown colonies on the SPamt medium. SPbc contained sucrose and peptone and was supplemented with cephalexin and boric acid as antibiotics and Psm and Pca formed semi-translucent to white colonies on the SPbc medium. SPamt and SPbc suppressed the growth of several plant-associated bacteria (except the P . syringae complex). The growth of saprophytic bacteria in seeds on the different media was compared with that on King’s B medium, using five types of commercially available Raphanus sativus seeds. The suppression rate of DTarTA was 85–99% and was lower for seeds with more saprophytic bacteria. The suppression rates of SPamt and SPbc were 90–99%. In detection tests using 10,000 seed samples mixed with Pca or Psm -contaminated seeds, it was possible to selectively isolate Psm and Pca using SPamt and SPbc, even when the colony numbers of the target bacterium constituted less than 10% of the total colonies. Key points • Bacterial leaf spot and blight pathogens were selectively isolated from seeds . • DTarTA medium distinguishes these pathogens from P. syringae complex pathovars . • SPamp and SPbc media have different selectivity for plant-associated bacteria .

Arabidopsis (Arabidopsis thaliana) NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1), a plasma membrane-localized protein, plays an essential role in resistance mediated by the coiled-coil-nucleotide-binding site-leucine-rich repeat class of resistance (R) proteins, which includes RESISTANCE TO PSEUDOMONAS SYRINGAE2 (RPS2), RESISTANCE TO PSEUDOMONAS SYRINGAE PV MACULICOLAl, and RPS5. Infection with Pseudomonas syringae pv tomato DC3000 expressing the bacterial effector proteins AvrRpt2, AvrB, and AvrPphB activates resistance by the aforementioned R proteins. Whereas the genetic requirement for NDR1 in plant disease resistance signaling has been detailed, our study focuses on determining a global, physiological role for NDR1. Through the use of homology modeling and structure threading, NDR1 was predicted to have a high degree of structural similarity to Arabidopsis LATE EMBRYOGENESIS ABUNDANT14, a protein implicated in abiotic stress responses. Specific protein motifs also point to a degree of homology with mammalian integrins, wellcharacterized proteins involved in adhesion and signaling. This structural homology led us to examine a physiological role for NDR1 in preventing fluid loss and maintaining cell integrity through plasma membrane-cell wall adhesions. Our results show a substantial alteration in induced (i.e. pathogen-inoculated) electrolyte leakage and a compromised pathogen-associated molecular pattern-triggered immune response in ndrl-1 mutant plants. As an extension of these analyses, using a combination of genetic and cell biology-based approaches, we have identified a role for NDRl in mediating plasma membrane-cell wall adhesions. Taken together, our data point to a broad role for NDR1 both in mediating primary cellular functions in Arabidopsis through maintaining the integrity of the cell wall-plasma membrane connection and as a key signaling component of these responses during pathogen infection.

Plants deploy intracellular innate immune receptors to recognize pathogens and initiate disease resistance. These nucleotide-binding, leucine-rich repeat (NB-LRR) proteins are activated by pathogen effector proteins that are delivered into the host cell to suppress host defense responses. Little is known about the sites and mechanisms of NB-LRR activation, but some NB-LRR proteins can function inside the plant nucleus. We demonstrate that RPM1 is activated on the plasma membrane and does not relocalize to the nucleus. An autoactive RPM1(D505V) allele that recapitulates key features of normal RPM1 activation also resides on the plasma membrane. There is no detectable relocalization of activated RPM1 to the nucleus. Hindering potential nuclear entry of RPM1-Myc did not affect either its effector-triggered hypersensitive-response (HR) cell death or its disease resistance functions, further suggesting that nuclear translocation is not required for RPM1 function. RPM1 tethered onto the plasma membrane with a dual acylated N-terminal epitope tag retained the ability to mediate HR, consistent with this RPM1 function being activated on the plasma membrane. Plant NB-LRR proteins can thus function at various locations in the cell.

In the absence of adaptive immunity displayed by animals, plants respond locally to biotic challenge via inducible basal defense networks activated through recognition and response to conserved pathogen-associated molecular patterns. In addition, immunity can be induced in tissues remote from infection sites by systemic acquired resistance (SAR), initiated after gene-for-gene recognition between plant resistance proteins and microbial effectors. The nature of the mobile signal and remotely activated networks responsible for establishing SAR remain unclear. Salicylic acid (SA) participates in the local and systemic response, but SAR does not require long-distance translocation of SA. Here, we show that, despite the absence of pathogen-associated molecular pattern contact, systemically responding leaves rapidly activate a SAR transcriptional signature with strong similarity to local basal defense. We present several lines of evidence that suggest jasmonates are central to systemic defense, possibly acting as the initiating signal for classic SAR. Jasmonic acid (JA), but not SA, rapidly accumulates in phloem exudates of leaves challenged with an avirulent strain of Pseudomonas syringae. In systemically responding leaves, transcripts associated with jasmonate biosynthesis are up-regulated within 4 h, and JA increases transiently. SAR can be mimicked by foliar JA application and is abrogated in mutants impaired in jasmonate synthesis or response. We conclude that jasmonate signaling appears to mediate long-distance information transmission. Moreover, the systemic transcriptional response shares extraordinary overlap with local herbivory and wounding responses, indicating that jasmonates may be pivotal to an evolutionarily conserved signaling network that decodes multiple abiotic and biotic stress signals.

Key messageArabidopsis LONG-CHAIN BASE KINASE 1 (LCBK1) interacts with MEDEA, a component of PCR2 complex that negatively regulates immunity. LCBK1 phosphorylates phytosphingosine and thereby promotes stomatal immunity against bacterial pathogens.Arabidopsis polycomb-group repressor complex2 (PRC2) protein MEDEA (MEA) suppresses both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). MEA represses the expression of RPS2 and thereby attenuates AvrRpt2 effector-mediated ETI. However, the mechanism of MEA-mediated PTI diminution was not known. By screening the Arabidopsis cDNA library using yeast-2-hybrid interaction, we identified LONG-CHAIN BASE KINASE1 (LCBK1) as an MEA-interacting protein. We found that lcbk1 mutants are susceptible to virulent bacterial pathogens, such as Pseudomonas syringae pv maculicola (Psm) and P. syringae pv tomato (Pst) but not the avirulent strain of Pst that carries AvrRpt2 effector. Pathogen inoculation induces LCBK1 expression, especially in guard cells. We found that LCBK1 has a positive regulatory role in stomatal closure after pathogen inoculation. WT plants close stomata within an hour of Pst inoculation or flg22 (a 22 amino acid peptide from bacterial flagellin protein that activates PTI) treatment, but not lcbk1 mutants. LCBK1 phosphorylates phytosphingosine (PHS). Exogenous application of phosphorylated PHS (PHS-P) induces stomatal closure and rescues loss-of-PTI phenotype of lcbk1 mutant plants. MEA overexpressing (MEA-Oex) plants are defective, whereas loss-of-function mea-6 mutants are hyperactive in PTI-induced stomatal closure. Exogenous application of PHS-P rescues loss-of-PTI in MEA-Oex plants. Results altogether demonstrate that LCBK1 is an interactor of MEA that positively regulates PTI-induced stomatal closure in Arabidopsis.

Bacterial leaf spot and bacterial leaf blight are global threats to the cultivation of cruciferous vegetables, and it is necessary to develop methods to easily detect, identify, and distinguish the causative pathogens Pseudomonas syringae pv. maculicola (Psm) and P. cannabina pv. alisalensis (Pca). Here, we used the sequence specificity of the exchangeable effector loci flanking the hrp gene cluster to design primers that can help detect and discriminate between Psm and Pca. Primers common to both bacteria (hrpK_fw1 and hrpK_fw2) were designed within hrpK at the end of the hrp gene cluster. Psm-specific primers (MAC_rv1 and MAC_rv2) were designed in hopPtoB1 and Pca-specific primers (ALS_rv1 and ALS_rv2) were designed in hopX1 adjacent to hrpK. PCR using hrpK_fw1 and MAC_rv1 or hrpK_fw2 and MAC_rv2 amplified DNA fragments of only Psm, P. syringae pv. tomato (causal agent of tomato bacterial speck), and P. syringae pv. spinaciae (causal agent of spinach bacterial leaf spot), among 76 strains of phytopathogenic bacteria. PCR using hrpK_fw1 and ALS_rv1 or hrpK_2 and ALS_rv2 amplified DNA fragments of only Pca. Multiplex PCR with these primers could easily distinguish Psm and Pca from bacterial colonies isolated on growth media and detect the pathogen in symptomatic leaves. Multiplex nested PCR with the primers detected contamination in one Psm- and/or one Pca-infected seeds in 1000 seeds. These results suggest that these PCR primers could help detect and discriminate Psm and Pca.Key points• We investigated Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis.• Novel primers common to both bacteria were designed following genome comparison.• Multiplex PCR with new primers could discriminate Psm and Pca.