Plasma membrane-nucleo-cytoplasmic coordination of a receptor-like cytoplasmic kinase promotes EDS1-dependent plant immunity.

Plants use cell-surface immune receptors to recognize pathogen-specific patterns to evoke basal immunity. ENHANCED DISEASE SUSCEPTIBILITY (EDS1) is known to be crucial for plant basal immunity, whereas its activation mechanism by pattern recognition remains enigmatic. Here, we show that the fungal pattern chitin induced the plasma membrane-anchored receptor-like cytoplasmic kinase PBS1-LIKE 19 (PBL19) to undergo nuclear translocation in Arabidopsis. The palmitoylation-deficient PBL19C3A variant constantly resided in the nucleus, triggering transcriptional self-amplification mainly through WRKY8 and EDS1-dependent constitutive immunity. Unexpectedly, the metacaspase-cleaved PBL19 lacking the N-terminal nuclear localization sequence specifically interacted with and phosphorylated EDS1 in the cytoplasm. Phosphodeficient EDS1 attenuated PBL19C3A-induced constitutive immunity, while phosphomimetic EDS1 complemented the loss of PBL19 for fungal resistance. Collectively, these findings reveal a compelling model wherein the plasma membrane, nuclear and cytoplasmic pools of PBL19 temporally coordinate distinct roles of immune signal receiver, amplifier and effector to boost plant antifungal immunity via EDS1.

BAK1-mediated phosphorylation of canonical G protein alpha during flagellin signaling in Arabidopsis.

Heterotrimeric G proteins consisting of Gα, Gß and Gγ are conserved signaling hubs in eukaryotes. Without analogs to canonical animal G protein-coupled receptors, plant cells are thought to use RGS1 and a yet unknown mechanism to regulate the activity of Gα. Meanwhile, the exact role of canonical Gα in plant innate immunity remains controversial. Here, we report multiple immune deficiencies in the null allele of Arabidopsis Gα (GPA1) in response to bacterial flg22 elicitor, clarifying a positive regulatory role of GPA1 in flg22 signaling. We also detect overall increased phosphorylation of GPA1 but reduced phosphorylation at Thr19 upon flg22 elicitation. Interestingly, flg22 could not induce phosphorylation of GPA1T19A and GPA1T19D , suggesting that the dynamic Thr19 phosphorylation is required for GPA1 to respond to flg22. Moreover, flg22-induced GPA1 phosphorylation is largely abolished in the absence of BAK1 in vivo, and BAK1 could phosphorylate GPA1 but not GPA1T19A in vitro at the phosphorylation sites identified in vivo, suggesting BAK1 is likely the kinase for GPA1 phosphorylation in response to flg22. Furthermore, the T19A mutation could promote flg22-induced association, rather than dissociation, between GPA1 and RGS1. Taken together, our findings shed new insights into the function and regulation of GPA1 in Arabidopsis defense signaling.

Enhanced protoplast assay by transfecting PCR-assembled gene expression cassettes with telomeric repeats and thiophosphate modifications.

Transient expression assays are invaluable complements to the stable transgenic assay for studying gene functions by providing desirable time and labor efficiencies and high-throughput potential or circumventing technical difficulties of stable transgenic expression. The protoplast transient expression system is one of the mainstream transient expression assays used in plant research. Here, we developed a PCR amplicon-mediated protoplast transient (PROMPT) assay by using overlapping PCR assembled gene expression cassettes for Arabidopsis protoplast transfection without the need for time- and labor-consuming plasmid construction. When 200 µl of Arabidopsis protoplasts were transfected with 1 µg of PCR amplicons or plasmid DNA, we detected substantially higher gene expression in the former. Moreover, we found that adding telomeric repeats and thiophosphate modifications to the 5' end of the nonsense strand through the reverse primer could further increase the PCR amplicon-mediated gene expression in protoplasts. Importantly, these improvements could also be applied to the protoplast assays in other dicot and monocot species including tobacco, rice and wheat. In addition, the subcellular localization of immune receptor FLS2 could be analyzed by PROMPT method. The PROMPT assay allows an accelerated and robust transient gene expression in protoplasts from diverse plant species.

Impact of IL-8-251A/T gene polymorphism on severity of disease caused by enterovirus 71 infection.

The study was performed in EV71-infected patients, with 97 mild cases and 80 severe cases. IL-8251A/T genotypes were determined by the polymerase chain reaction restriction fragment length polymorphism technique. Severe cases had a significantly higher frequency of the IL8-251 AT and TT genotypes than mild cases (52.5 % vs. 49.5 % and 42.5 % vs. 30.9 %, respectively; p = 0.024). The frequency of IL-8-251T alleles among the severe cases was also significantly higher than that of mild cases (68.7 % vs. 55.7 %, OR = 1.8, 95 % CI = 1.1-2.7, p = 0.012). There were significant differences in gender, age, fever days, WBC, CRP and BG concentration, and IL-8 levels among genotypes of IL-8251A/T in EV71-infected patients, but there were no significant differences in ALT, AST, CK-MB and EV71 loads. These findings suggested that the IL-8-251T allele is associated with susceptibility to severe disease in Chinese patients infected with EV71.

[Occult HBV infection in patients with anti-HBc positive alone].

OBJECTIVE: This study was designed to explore the incidence rate of occult HBV infection in patients with anti-HBc positive alone and analyze the possible reasons of occult infection. METHODS: Sera of 183 patients carrying anti-HBc alone(A < or = 0.1) were collected and real-time PCR was used to select samples with HBV DNA positive. HBV pre-S/S amplification products were obtained by PCR, and clonal sequencing were then used for these samples with HBV DNA positive. RESULTS: DNA quantitative results of three samples were greater than 10(3) copies/ml in 183 samples, with a fraction of 1.6%. Pre-S/S sequencing results of two samples from these three samples were obtained. Point mutations within "a" determinant with Q129R/P mutations and co-existence of the mutant type and wild type were found in the two samples. CONCLUSIONS: Occult HBV infection existed in samples with anti-HBc alone. Factors contributing to the loss of HBsAg detection by immunoassays include S gene mutations and low levels of circulating antigen which are below the assay limit of detection. Occult HBV infection not only can lead to a false clinical diagnosis, but also can result in hematological pollution due to such occult infection of blood donors.