The receptor-like kinase SERK3/BAK1 is required for basal resistance against the late blight pathogen phytophthora infestans in Nicotiana benthamiana

Background

The filamentous oomycete plant pathogen Phytophthora infestans causes late blight, an economically important disease, on members of the nightshade family (Solanaceae), such as the crop plants potato and tomato. The related plant Nicotiana benthamiana is a model system to study plant-pathogen interactions, and the susceptibility of N. benthamiana to Phytophthora species varies from susceptible to resistant. Little is known about the extent to which plant basal immunity, mediated by membrane receptors that recognise conserved pathogen-associated molecular patterns (PAMPs), contributes to P. infestans resistance.

Principal Findings

We found that different species of Phytophthora have varying degrees of virulence on N. benthamiana ranging from avirulence (incompatible interaction) to moderate virulence through to full aggressiveness. The leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1/SERK3 is a major modulator of PAMP-triggered immunity (PTI) in Arabidopsis thaliana and N. benthamiana. We cloned two NbSerk3 homologs, NbSerk3A and NbSerk3B, from N. benthamiana based on sequence similarity to the A. thaliana gene. N. benthamiana plants silenced for NbSerk3 showed markedly enhanced susceptibility to P. infestans infection but were not altered in resistance to Phytophthora mirabilis, a sister species of P. infestans that specializes on a different host plant. Furthermore, silencing of NbSerk3 reduced the cell death response triggered by the INF1, a secreted P. infestans protein with features of PAMPs.

Conclusions/Significance

We demonstrated that N. benthamiana NbSERK3 significantly contributes to resistance to P. infestans and regulates the immune responses triggered by the P. infestans PAMP protein INF1. In the future, the identification of novel surface receptors that associate with NbSERK3A and/or NbSERK3B should lead to the identification of new receptors that mediate recognition of oomycete PAMPs, such as INF1.     

The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana

The RXLR cytoplasmic effector AVR3a of Phytophthora infestans confers avirulence on potato plants carrying the R3a gene. Two alleles of Avr3a encode secreted proteins that differ in only three amino acid residues, two of which are in the mature protein. Avirulent isolates carry the Avr3a allele, which encodes AVR3aKI (containing amino acids C19, K80 and I103), whereas virulent isolates express only the virulence allele avr3a, encoding AVR3aEM (S19, E80 and M103). Only the AVR3aKI protein is recognized inside the plant cytoplasm where it triggers R3a-mediated hypersensitivity. Similar to other oomycete avirulence proteins, AVR3aKI carries a signal peptide followed by a conserved motif centered on the consensus RXLR sequence that is functionally similar to a host cell-targeting signal of malaria parasites. The interaction between Avr3a and R3a can be reconstructed by their transient co-expression in Nicotiana benthamiana. We exploited the N. benthamiana experimental system to further characterize the Avr3a-R3a interaction. R3a activation by AVR3aKI is dependent on the ubiquitin ligase-associated protein SGT1 and heat-shock protein HSP90. The AVR3aKI and AVR3aEM proteins are equally stable in planta, suggesting that the difference in R3a-mediated death cannot be attributed to AVR3aEM protein instability. AVR3aKI is able to suppress cell death induced by the elicitin INF1 of P. infestans, suggesting a possible virulence function for this protein. Structure-function experiments indicated that the 75-amino acid C-terminal half of AVR3aKI, which excludes the RXLR region, is sufficient for avirulence and suppression functions, consistent with the view that the N-terminal region of AVR3aKI and other RXLR effectors is involved in secretion and targeting but is not required for effector activity. We also found that both polymorphic amino acids, K80 and I103, of mature AVR3a contribute to the effector functions.     

Synergistic interactions of the plant cell death pathways induced by Phytophthora infestans Nepl-like protein PiNPP1.1 and INF1 elicitin

Cell death plays a ubiquitous role in plant-microbe interactions, given that it is associated with both susceptible and resistance interactions. A class of cell death-inducing proteins, termed Nepl-like proteins (NLPs), has been reported in bacteria, fungi, and oomycetes. These proteins induce nonspecific necrosis in a variety of dicotyledonous plants. Here, we describe three members of the NLP family from the oomycete Phytophthora infestans (PiNPP1.1, PiNPP1.2, and PiNPP1.3). Using agroinfection with a binary Potato virus X vector, we showed that PiNPP1.1 induces cell death in Nicotiana benthamiana and the host plant tomato. Expression analyses indicated that PiNPP1.1 is up-regulated during late stages of infection of tomato by P. infestans. We compared PiNPP1.1 necrosis-inducing activity to INF1 elicitin, a well-studied protein that triggers the hypersensitive response in Nicotiana spp. Using virus-induced gene silencing, we showed that the cell death induced by PiNPP1.1 is dependent on the ubiquitin ligase-associated protein SGT1 and the heat-shock protein HSP90. In addition, cell death triggered by PiNPP1.1 but not that by INF1 was dependent on the defense-signaling proteins COI1, MEK2, NPR1, and TGA2.2, suggesting distinct signaling requirements. Combined expression of PiNPP1.1 and INF1 in N. benthamiana resulted in enhanced cell death, suggesting synergistic interplay between the two cell-death responses. Altogether, these results point to potentially distinct but interacting cell-death pathways induced by PiNPP1.1 and INF1 in plants.     

马铃薯晚疫病菌全基因组分泌蛋白的初步分析

利用马铃薯晚疫病菌全基因组测序结果,结合计算机技术和生物信息学的方法,对马铃薯晚疫病菌的蛋白进行分析,为明确该病原菌与寄主互作的分子机制奠定基础。文章应用信号肽预测软件SignalP v3.0和PSORT,跨膜螺旋结构预测软件TMHMM-2.0和THUMBUP,GPI锚定位点预测软件big-PI Predictor,亚细胞器中蛋白定位分布预测软件TargetP v1.01,对已经公布的马铃薯晚疫病菌全基因组22 658个蛋白质氨基酸序列进行分析。结果发现,晚疫病菌全基因组编码蛋白中有671个为潜在的分泌型蛋白,占编码蛋白总数的3.0%。其中有45个分泌蛋白有功能方面的描述,其功能涉及细胞代谢、信号转导等方面;此外,还有一些与激发子类似的分泌蛋白,它们可能与晚疫病菌的毒性有关。     

A pollen tube growth-promoting arabinogalactan protein from nicotiana alata is similar to the tobacco TTS protein

Upon germination on the stigma, pollen tubes elongate in the stylar transmitting tract, aided by female factors, with speed and directionality not mimicked in in vitro pollen tube growth cultures. We have shown that a stylar transmitting tissue arabinogalactan protein (AGP) from Nicotiana tabacum (tobacco), TTS protein, stimulates pollen tube growth in vivo and in vitro and attracts pollen tubes grown in a semi-in vivo culture system. It has been reported that the self-incompatible Nicotiana alata produced a stylar glycoprotein, GaRSGP, which had a backbone polypeptide that shared 97% identity with those of TTS proteins but some of its properties were different from those described for TTS proteins. We report here the characterization of a family of stylar transmitting tissue glycoproteins from N. alata that is virtually identical to tobacco TTS proteins and which we refer to as NaTTS proteins. Like their tobacco counterparts, NaTTS proteins are recognized by the traditional AGP-diagnostic reagent beta-glucosyl Yariv reagent, and they are also recognized by JIM13, a monoclonal antibody against AGP. NaTTS proteins also stimulate pollen tube elongation in vitro and attract pollen tubes in a semi-in vivo pollen tube culture system. Biochemical and immunological characterization of NaTTS proteins revealed that they have extraordinary variability in the extent of sugar modifications of their polypeptide backbones. The extent of sugar modifications on NaTTS proteins significantly affects their biochemical properties, influences how they interact with the transmitting tissue extracellular matrix, and affects their solubility from this matrix. Our results suggest that the strategy used to purify GaRSGP only recovered a less glycosylated, more tightly extracellular matrix-bound sub-population of the entire spectrum of N. alata TTS proteins.     

Stress fiber strain recognition by the LIM protein testin is cryptic and mediated by RhoA

The actin cytoskeleton is a key regulator of mechanical processes in cells. The family of LIM domain proteins have recently emerged as important mechanoresponsive cytoskeletal elements capable of sensing strain in the actin cytoskeleton. The mechanisms regulating this mechanosensitive behavior, however, remain poorly understood. Here we show that the LIM domain protein testin is peculiar in that despite the full-length protein primarily appearing diffuse in the cytoplasm, the C-terminal LIM domains alone recognize focal adhesions and strained actin, while the N-terminal domains alone recognize stress fibers. Phosphorylation mutations in the dimerization regions of testin, however, reveal its mechanosensitivity and cause it to relocate to focal adhesions and sites of strain in the actin cytoskeleton. Finally, we demonstrate that activated RhoA causes testin to adorn stress fibers and become mechanosensitive. Together, our data show that testin’s mechanoresponse is regulated in cells and provide new insights into LIM domain protein recognition of the actin cytoskeleton’s mechanical state.     

Degradation of human Aurora-A protein kinase is mediated by hCdh1

Human Aurora-A is related to a protein kinase originally identified by its close homology to Ipl1p from Saccharomyces cerevisiae and aurora from Drosophila melanogaster, which are key regulators of the structure and function of the mitotic spindle. We previously showed that human Aurora-A is turned over through the anaphase promoting complex/cyclosome (APC/C)-ubiquitin-proteasome pathway. The association of two distinct WD40 repeat proteins known as Cdc20 and Cdh1, respectively, sequentially activates the APC/C. The present study shows that Aurora-A degradation is dependent on hCdh1 in vivo, not on hCdc20, and that Aurora-A is targeted for proteolysis through distinct structural features of the destruction box, the KEN box motifs and its kinase activity.     

Resistance to Bt maize in Mythimna unipuncta (Lepidoptera: Noctuidae) is mediated by alteration in Cry1Ab protein activation

Bt maize cultivars based on the event MON810 (expressing Cry1Ab) have shown high efficacy for controlling corn borers. However, their efficiency for controlling some secondary lepidopteran pests such as Mythimna unipuncta has been questioned, raising concerns about potential outbreaks and its economic consequences. We have selected a resistant strain (MR) of M. unipuncta, which is capable of completing its life cycle on Bt maize and displays a similar performance when feeding on both Bt and non-Bt maize. The proteolytic activation of the protoxin and the binding of active toxin to brush border membrane vesicles were investigated in the resistant and a control strain. A reduction in the activity of proteolytic enzymes, which correlates with impaired capacity of midgut extracts to activate the Cry1Ab protoxin has been observed in the resistant strain. Moreover, resistance in larvae of the MR strain was reverted when treated with Cry1Ab toxin activated with midgut juice from the control strain. All these data indicate that resistance in the MR strain is mediated by alteration of toxin activation rather than to an increase in the proteolytic degradation of the protein. By contrast, binding assays performed with biotin labelled Cry1Ab suggest that binding to midgut receptors does not play a major role in the resistance to Bt maize. Our results emphasize the risk of development of resistance in field populations of M. unipuncta and the need to consider this secondary pest in ongoing resistance management programs to avoid the likely negative agronomic and environmental consequences.     

Targeting of the ring exported protein 1 to the Maurer's clefts is mediated by a two-phase process

Early development of Plasmodium falciparum within the erythrocyte is characterized by the large-scale export of proteins to the host cell. In many cases, export is mediated by a short sequence called the Plasmodium export element (PEXEL) or vacuolar transport signal; however, a number of previously characterized exported proteins do not contain such an element. In this study, we investigated the mechanisms of export of the PEXEL-negative ring exported protein 1 (REX1). This protein localizes to the Maurer's clefts, parasite-induced structures in the host-cell cytosol. Transgenic parasites expressing green fluorescent protein–REX1 chimeras revealed that the single hydrophobic stretch plus an additional 10 amino acids mediate the export of REX1. Biochemical characterization of these chimeras indicated that REX1 was exported as a soluble protein. Inclusion of a sequence containing a predicted coiled-coil motif led to the correct localization of REX1 at the Maurer's clefts, suggesting that association with the clefts occurs at the final stage of protein export only. These results indicate that PEXEL-negative exported proteins can be exported in a soluble state and that sequences without any apparent resemblance to a PEXEL motif can mediate export across the parasitophorous vacuole membrane.     

Coat protein mediated resistance to Plum Pox Virus in Nicotiana clevelandii and N. benthamiana

Transgenic Nicotiana benthamiana and N. clevelandii plants expressing the coat protein of Plum Pox Virus under the control of the 35S promoter from Cauliflower Mosaic Virus were engineered by Agrobacterium tumefaciens mediated transformation. The phenomenon of virus resistance was observed at different levels when transgenic plants, expressing the coat protein and control plants were compared after challenge infection with Plum Pox Virus. N. clevelandii coat protein transgenic plants circumvent virus accumulation. After an initial increase in virus titer similar to the control plants, some coat protein expressing plants showed a reduced accumulation of virus and inhibition of the systemic spread, characterized by decrease of the virus titer and formation of new symptomless leaves. In other N. clevelandii coat protein expressing plants virus accumulation was inhibited and disease symptoms never appeared. N. benthamiana coat protein expressing plants were also protected. After a temporary virus accumulation, virus titer decreased without the appearance of symptoms with the exception of a few plants, which showed a delay of thirty days in the development of symptoms post challenge infection.Abbreviations PPV Plum Pox Virus - CP coat protein - CaMV Cauliflower Mosaic Virus - CP+ coat protein expressing plant - CP– control plant = non coat protein expressing plant - TMV Tobacco Mosaic Virus - NPTII neomycin phosphotransferaseII - IBA indole-3-butyric acid - BAP 6-benzylaminopurine; - MS Murashige Skoog - ELISA enzyme linked immunosorbent assay     

Arsenate reduction mediated by the plasmid-encoded ArsC protein is coupled to glutathione

Resistance to arsenate conferred on Escherichia coli by the ars operon of plasmid R773 requires both the product of the arsC gene and reduction of arsenate to arsenate. A genetic analysis was performed to identify the source of reducing potential in vivo. in addition to the ars genes, arsenate resistance required the products of the gor gene for glutathione reductase and the gshA and gshB genes for glutathione synthesis. Mutations in the trx and grx genes for thioredoxin and glutaredoxin, respectively, had no effect on arsenate resistance. Although resistance required the arsC gene, the rate of reduction of arsenate to arsenate was nearly the same in cells lacking the ars operon. In strains deficient in glutathione biosynthesis this endogenous reduction was greatly diminished, and cells exhibited increased sensitivity to arsenate. When glutathione was supplied exogenously to such mutants, resistance was restored only to cells expressing the ars operon, and only such cells had detectable arsenate reduction after addition of glutathione. Since ArsC-catalysed reduction of arsenate provides high level resistance, physical coupling of the ArsC reaction to efflux of the resulting arsenite is hypothesised.     

The prokaryotic activity of the IGR IRESs is mediated by ribosomal protein S1

Internal ribosome entry sites (IRESs) are RNA elements capable of initiating translation on an internal portion of a messenger RNA. The intergenic region (IGR) IRES of the Dicistroviridae virus family folds into a triple pseudoknot tertiary structure, allowing it to recruit the ribosome and initiate translation in a structure dependent manner. This IRES has also been reported to drive translation in Escherichia coli and to date is the only described translation initiation signal that functions across domains of life. Here we show that unlike in the eukaryotic context the tertiary structure of the IGR IRES is not required for prokaryotic ribosome recruitment. In E. coli IGR IRES translation efficiency is dependent on ribosomal protein S1 in conjunction with an AG-rich Shine-Dalgarno-like element, supporting a model where the translational activity of the IGR IRESs is due to S1-mediated canonical prokaryotic translation.     

IFNbeta induction by influenza A virus is mediated by RIG-I which is regulated by the viral NS1 protein

Influenza A virus causes epidemics of respiratory diseases in humans leading to thousands of death annually. One of its major virulence factors, the non-structural protein 1 (NS1), exhibits interferon-antagonistic properties. While epithelial cells of the respiratory tract are the primary targets of influenza virus, the virus-sensing mechanisms in these cells eventually leading to IFNbeta production are incompletely understood. Here we show that infection of epithelial cells with NS1-deficient influenza A virus upregulated expression of two molecules that have been previously implicated in sensing of RNA viruses, the retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene 5 (MDA5). Gene silencing and overexpression experiments demonstrated that RIG-I, its adapter interferon-beta promoter stimulator 1 (IPS-1) and interferon-regulated factor 3 (IRF3) were involved in influenza A virus-mediated production of the antiviral IFNbeta. In addition, we showed that the NS1 protein is capable to inhibit the RIG-I-induced signalling, a mechanism which corresponded to the observation that only NS1-deficient but not the wild-type virus induced high-level production of IFNbeta. In conclusion, we demonstrated a critical involvement of RIG-I, IPS-1 and IRF3 in influenza A virus infection of epithelial cells.     

Degradation of misfolded protein in the cytoplasm is mediated by the ubiquitin ligase Ubr1

Protein quality control and subsequent elimination of terminally misfolded proteins occurs via the ubiquitin-proteasome system. Tagging of misfolded proteins with ubiquitin for degradation depends on a cascade of reactions involving an ubiquitin activating enzyme (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). While ubiquitin ligases responsible for targeting misfolded secretory proteins to proteasomal degradation (ERAD) have been uncovered, no such E3 enzymes have been found for elimination of misfolded cytoplasmic proteins in yeast. Here we report on the discovery of Ubr1, the E3 ligase of the N-end rule pathway, to be responsible for targeting misfolded cytosoplasmic protein to proteasomal degradation.