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Activation of nucleotide-binding leucine-rich repeat receptors (NLRs) results in immunity and a localized cell death. NLR cell death activity requires oligomerization and in some cases plasma membrane (PM) localization. The exact mechanisms underlying PM localization of NLRs lacking predicted transmembrane domains or recognizable lipidation motifs remain elusive.We used confocal microscopy, genetically encoded molecular tools and protein-lipid overlay assays to determine whether PM localization of members of the Arabidopsis HeLo-/RPW8-like domain ‘helper’ NLR (RNL) family is mediated by the interaction with negatively charged phospholipids of the PM.Our results show that PM localization and stability of some RNLs and one CC-type NLR (CNL) depend on the direct interaction with PM phospholipids. Depletion of phosphatidylinositol-4-phosphate from the PM led to a mis-localization of the analyzed NLRs and consequently inhibited their cell death activity. We further demonstrate homo- and hetero-association of members of the RNL family. Our results provide new insights into the molecular mechanism of NLR localization and defines an important role of phospholipids for CNL and RNL PM localization and consequently, for their function.We propose that RNLs interact with anionic PM phospholipids and that RNL-mediated cell death and immune responses happen at the PM

With background concentrations having reached phytotoxic levels during the last century, tropospheric ozone (O 3 ) has become a key climate change agent, counteracting carbon sequestration by forest ecosystems. One of the main knowledge gaps for implementing the recent O 3 flux-based critical levels (CLs) concerns the assessment of effective O 3 dose leading to adverse effects in plants. In this study, we investigate the dynamics of physiological, structural, and morphological responses induced by two levels of O 3 exposure (80 and 100 ppb) in the foliage of hybrid poplar, as a function of phytotoxic O 3 dose (POD 0 ) and foliar developmental stage. After a latency period driven by foliar ontological development, the gas exchanges and chlorophyll content decreased with higher POD 0 monotonically. Hypersensitive response-like lesions appeared early during exposure and showed sigmoidal-like dynamics, varying according to leaf age. At current POD 1_SPEC CL, notwithstanding the aforementioned reactions and initial visible injury to foliage, the treated poplars had still not shown any growth or biomass reduction. Hence, this study demonstrates the development of a complex syndrome of early reactions below the flux-based CL, with response dynamics closely determined by the foliar ontological stage and environmental conditions. General agreement with patterns observed in the field appears indicative of early O 3 impacts on processes relevant, e.g., biodiversity ecosystem services before those of economic significance – i.e., wood production, as targeted by flux-based CL.

Plant responses to enemies are coordinated by several interacting signaling systems. Molecular and genetic studies with mutants and exogenous signal application suggest that jasmonate (JA)-, salicylate (SA)-, and ethylene (ET)-mediated pathways modulate expression of portions of the defense phenotype in Arabidopsis (Arabidopsis thaliana), but have not yet linked these observations directly with plant responses to insect attack. We compared the glucosinolate (GS) profiles of rosette leaves of 4-week-old mutant and transgenic Arabidopsis (Columbia) plants compromised in these three major signaling pathways, and characterized responses by those plants to feeding by two phloem-feeding aphids (generalist Myzus persicae and specialist Brevicoryne brassicae) and one generalist caterpillar species (Spodoptera exigua Hubner). Blocked JA signaling in coronatine-insensitive (coi1) and enhanced expression of SA-signaled disease resistance in hypersensitive response-like (hrl1) mutants reduced constitutive GS concentrations, while blocking SA signaling at the mediator protein npr1 mutant (NPR) increased them. There was no significant impact on constitutive GS contents of blocking ET signaling (at ET resistant [etr1]) or reducing SA concentrations (nahG transgene). We found increased GS accumulation in response to insect feeding, which required functional NPR1 and ETR1 but not COI1 or SA. Insect feeding caused increases primarily in short-chain aliphatic methylsulfinyl GS. By contrast, responses to exogenous JA, a frequent experimental surrogate for insect attack, were characterized by an increase in indolyl GS. Insect performance, measured as population increase or weight increase, was negatively related to GS levels, but we found evidence that other, ET-regulated factors may also be influential. Plant resistance to (consumption by) S. exigua was not related to insect growth because some plant chemistries inhibited growth while others inhibited feeding. These major signaling pathways modulate Arabidopsis GS accumulation and response to both phloem-feeding and chewing insects, often antagonistically; NPR appears to be central to these interactions. Our results indicate that exogenous signal application and plant consumption measures may not provide useful measures of plant responses to actual insect feeding.

Soybean mosaic virus (SMV) is one of the most prevalent and important pathogens of soybean, which produces 11 proteins, and the third protein, P3, was suggested to be involved in virus movement and replication, as well as host infection. During the virus infection, host proteins are essential in the virus cycle. However, there is no comprehensive report on the network of host proteins that interact with P3. Fifty-one interactors were identified by using the P3 protein as the bait against the SMV SC15 strain-challenged soybean cDNA library. These proteins were classified into five groups, including transport and protein transport-related proteins, defense and disease-related proteins, photosynthesis proteins, cellular metabolic proteins, and unknown proteins. Among these proteins, the protein defined as hypersensitive response-like lesion-inducing (HRLI) appeared multiple times and showed strong affinity with P3, which indicated its important role in SMV infection. Thus, it was chosen for further investigation. Phylogenetic classification showed that paralog proteins GmHRLI-1 and GmHRLI-2 clustered together and shared 90% homologous identity. Bimolecular fluorescence complementation (BiFC) assay was carried out to confirm the interaction, and fluorescence was detected at the cell periplasmic as well as at the nucleus. Subcellular localization showed that GmHRLI was localized to the cell periplasmic, while the co-localization of GmHRLI and P3 signals was also observed in the nucleus, suggesting that GmHRLI could interact with P3 and promoted the translation of P3 to the nucleus. Moreover, the gene expression of GmHRLI was abundant in the roots, leaves, and flowers, and could be induced by SMV infection, suggesting its involvement in SMV infection. Our results together lay the foundation to explore the mechanisms of P3 in the HR process and the HRLI protein function in SMV response.

Geminivirus beet curly top Iran virus (BCTIV) is one of the main causal agents of the beet curly top disease in Iran and the newly established Becurtovirus genus type species. Although the biological features of known becurtoviruses are similar to those of curtoviruses, they only share a limited sequence identity, and no information is available on the function of their viral genes. In this work, we demonstrate that BCTIV V2, as the curtoviral V2, is also a local silencing suppressor in Nicotiana benthamiana and can delay the systemic silencing spreading, although it cannot block the cell-to-cell movement of the silencing signal to adjacent cells. BCTIV V2 shows the same subcellular localization as curtoviral V2, being detected in the nucleus and perinuclear region, and its ectopic expression from a PVX-derived vector also causes the induction of necrotic lesions in N. benthamiana, such as the ones produced during the HR, both at the local and systemic levels. The results from the infection of N. benthamiana with a V2 BCTIV mutant showed that V2 is required for systemic infection, but not for viral replication, in a local infection. Considering all these results, we can conclude that BCTIV V2 is a functional homologue of curtoviral V2 and plays a crucial role in viral pathogenicity and systemic movement.

Ninety-six strains of Pantoea ananatis were isolated from 14 plant species including melon, rice, tea and other crops of economic importance. They were classified into three groups (group I, II, III) based on a welsh onion stabbing assay, tobacco infiltration test, and polymerase chain reaction to detect indole acetic acid (IAA) biosynthesis genes (iaaM and iaaH) and a cytokinin biosynthesis gene (etz). Group Ι strains were characterized as causing significant blight symptom on welsh onion and inducing a hypersensitive response (HR)-like reaction on tobacco leaves after 36-48 h and encompassed 20 isolates from foxtail millet, hydrangea, pineapple, river water and rice. These 20 isolates did not possess iaaM, iaaH, or etz genes. Group II, consisting of 34 melon isolates, harbored iaaM, iaaH and etz genes, but did not cause either blight on welsh onion or HR-like reaction on tobacco. Group III strains did not have the iaaM, iaaH, and etz genes, nor did they cause any reaction on welsh onion or tobacco. The 42 strains in group III were isolated from bamboo grass, Chinese silver grass, citrus, dogwood, melon, mugwort, silk tree, sweet corn, tea and welsh onion. Representative strains of the three groups were tested for pathogenicity on melon and rice. Group Ι strains caused palea browning on rice but not internal fruit rot on melon. On the contrary, group II strains did not cause disease on rice but caused internal fruit rot on melon. Group III strains were not pathogenic on rice or melon. These results suggested that the host range of P. ananatis may be predicted by the reactions of welsh onion and tobacco and detection of iaaM, iaaH and etz genes. These tools may serve as rapid tests to identify the pathogenicity groups of P. ananatis.

Aggressive reactive oxygen species (ROS) play an important role in plant defence against biotic stressors, including herbivorous insects. Plants may even generate ROS in response to insect eggs, thus effectively fighting against future larval herbivory. However, so far nothing is known on how ROS-mediated plant defence against insect eggs is enzymatically regulated. Neither do we know how insects cope with egg-induced plant ROS. We addressed these gaps of knowledge by studying the activities of ROS-related enzymes in Pinus sylvestris deposited with eggs of the herbivorous sawfly Diprion pini. This species cuts a slit into pine needles and inserts its eggs into the needle tissue. About a quarter of egg-deposited needles show chlorotic tissue at the oviposition sites, indicating hypersensitive response-like direct defence responses resulting in reduced larval hatching from eggs. Hydrogen peroxide and peroxidase sensitive staining of sections of egg-deposited pine needles revealed the presence of hydrogen peroxide and peroxidase activity in needle tissue close to the eggs. Activity of ROS-producing NADPH-oxidase did not increase after egg deposition. However, the activity of the ROS-detoxifying enzyme catalase decreased after egg deposition and ovipositional wounding of needles. These results show that local ROS accumulation at the oviposition site is not caused by increased NADPH-oxidase activity, but reduced activity of pine needle catalase may contribute to it. However, our data suggest that pine sawflies can counteract the egg deposition-induced hydrogen peroxide accumulation in pine needles by high catalase activity in their oviduct secretion which is released with the eggs into pine tissue.

. is the primary source of sugar and plays a significant role in global renewable bioenergy. (SCBV) is one of the most important viruses infecting sugarcane, causing severe yield losses and quality degradation. It is of great significance to reveal the pathogenesis of SCBV and resistance breeding. However, little is known about the viral virulence factors or RNA silencing suppressors and the molecular mechanism of pathogenesis. To systematically investigate the functions of the unknown protein P2 encoded by SCBV ORF2. Phylogenetic analysis was implemented to infer the evolutionary relationship between the P2 of SCBV and other badnaviruses. The precise subcellular localization of P2 was verified in the transient infiltrated epidermal mesophyll cells and protoplasts using the Laser scanning confocal microscope (LSCM). The post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) RNA silencing suppressor activity of P2 was analyzed, respectively. Furthermore, restriction digestion and RT-qPCR assays were conducted to verify the probable mechanism of P2 on repressing DNA methylation. To explore the pathogenicity of P2, a potato virus X-based viral vector was used to heterologously express SCBV P2 and the consequent H O accumulation was detected by the 3,3'-diaminobenzidine (DAB) staining method. Phylogenetic analysis shows that SCBV has no obvious sequence similarity and low genetic relatedness to and representatives. LSCM studies show that P2 is localized in both the cytoplasm and nucleus. Moreover, P2 is shown to be a suppressor of PTGS and TGS, which can not only repress ssRNA-induced gene silencing but also disrupt the host RNA-directed DNA methylation (RdDM) pathway. In addition, P2 can trigger an oxidative burst and cause typical hypersensitive-like response (HLR) necrosis in systemic leaves of when expressed by PVX. Overall, our results laid a foundation for deciphering the molecular mechanism of SCBV pathogenesis and made progress for resistance breeding.

Root-knot nematodes (RKN) are obligate parasite species of the genus Meloidogyne that cause great losses in Arabica coffee (Coffea arabica L.) plantations. Identification of resistant genotypes would facilitate the improvement of coffee varieties aiming at an environmental friendly and costless nematode control. In this work, the C. arabica genotype ‘UFV 408-28' was found to be resistant to the most destructive RKN species M. incognita. Pathogenicity assays indicated that the highly aggressive populations of M. incognita races 1, 2 and 3 were not able to successfully reproduce on ‘UFV 408-28' roots and displayed a low gall index (GI = 2). An average reduction of 87% reduction of the M. incognita population was observed on ‘UFV 408-28' when compared to the susceptible cultivar ‘IAC 15'. By contrast, ‘UFV 408-28' was susceptible to the related species M. exigua and M. paranaensis (GI = 5 and 4, respectively). Histological observations performed on sections of UFV408-28 roots infected with M. incognita race 1 showed that nematode infection could be blocked right after penetration or during migration and establishment stages, at 6 days, 7 days and 8 days after infection (DAI). Fluorescence and bright field microscopy observations showed that root cells surrounding the nematodes exhibited HR-like features such as accumulation of phenolic compounds and a necrotic cell aspect. In the susceptible ‘IAC 15' roots, 6 DAI, feeding sites contained giant cells with a dense cytoplasm. Necrotic cells were never observed throughout the entire infection cycle. The HR-like phenotype observed in the ‘UFV 408-28'—M. incognita interaction suggests that the coffee resistance may be mediated by a R-gene based immunity system and may therefore provide new insights for understanding the molecular basis of RKN resistance in perennial crops.

The proteolytic activity present in the extracellular preparation (ECP) from suspension media of infective structures of the late blight pathogen Phytophthora infestans was partially characterized. The proteolytic activity was analyzed in gelatin-containing SDS-PAGE. A discrete band of digested gelatin was visualized at approximately 45 kDa in ECPs from zoospores and germinating cysts media. Treatment of ECP with the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) or incubation at 100°C for 5 min completely abolished the proteolytic activity in the zymograph assay. When microinjected in potato leaves, ECP induce localized necrosis within 24 h post inoculation. This necrosis appeared in potato and was not visible in two non-host plants. Moreover, the necrosis seems to be dependent on active host metabolism. Treatments of ECP with Proteinase K, PMSF and boiling inhibited their ability to induce the necrotic response. These results suggest a correlation between, proteolytic and necrosis-inducing activities in ECP. A preliminary characterization with protease inhibitors suggests that the ECPs contain serine protease(s).[PUBLICATION ABSTRACT]