Chloroplasts alter their morphology and accumulate at the pathogen interface during infection by Phytophthora infestans.

Upon immune activation, chloroplasts switch off photosynthesis, produce antimicrobial compounds and associate with the nucleus through tubular extensions called stromules. Although it is well established that chloroplasts alter their position in response to light, little is known about the dynamics of chloroplast movement in response to pathogen attack. Here, we report that during infection with the Irish potato famine pathogen Phytophthora infestans, chloroplasts accumulate at the pathogen interface, associating with the specialized membrane that engulfs the pathogen haustorium. The chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at pathogen haustoria, suggesting that this process is partially dependent on the actin cytoskeleton. However, chloroplast accumulation at haustoria does not necessarily rely on movement of the nucleus to this interface and is not affected by light conditions. Stromules are typically induced during infection, embracing haustoria and facilitating chloroplast interactions, to form dynamic organelle clusters. We found that infection-triggered stromule formation relies on BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1)-mediated surface immune signaling, whereas chloroplast repositioning towards haustoria does not. Consistent with the defense-related induction of stromules, effector-mediated suppression of BAK1-mediated immune signaling reduced stromule formation during infection. On the other hand, immune recognition of the same effector stimulated stromules, presumably via a different pathway. These findings implicate chloroplasts in a polarized response upon pathogen attack and point to more complex functions of these organelles in plant-pathogen interactions.

Expression of pathogenesis-related proteins in transplastomic tobacco plants confers resistance to filamentous pathogens under field trials.

Plants are continuously challenged by pathogens, affecting most staple crops compromising food security. They have evolved different mechanisms to counterattack pathogen infection, including the accumulation of pathogenesis-related (PR) proteins. These proteins have been implicated in active defense, and their overexpression has led to enhanced resistance in nuclear transgenic plants, although in many cases constitutive expression resulted in lesion-mimic phenotypes. We decided to evaluate plastid transformation as an alternative to overcome limitations observed for nuclear transgenic technologies. The advantages include the possibilities to express polycistronic RNAs, to obtain higher protein expression levels, and the impeded gene flow due to the maternal inheritance of the plastome. We transformed Nicotiana tabacum plastids to co-express the tobacco PR proteins AP24 and ß-1,3-glucanase. Transplastomic tobacco lines were characterized and subsequently challenged with Rhizoctonia solani, Peronospora hyoscyami f.sp. tabacina and Phytophthora nicotianae. Results showed that transplastomic plants expressing AP24 and ß-1,3-glucanase are resistant to R. solani in greenhouse conditions and, furthermore, they are protected against P.hyoscyami f.sp. tabacina and P. nicotianae in field conditions under high inoculum pressure. Our results suggest that plastid co- expression of PR proteins AP24 and ß-1,3-glucanase resulted in enhanced resistance against filamentous pathogens.

Analysis of the potato calcium-dependent protein kinase family and characterization of StCDPK7, a member induced upon infection with Phytophthora infestans.

KEY MESSAGE: We describe the potato CDPK family and place StCDPK7 as a player in potato response to Phytophthora infestans infection, identifying phenylalanine ammonia lyase as its specific phosphorylation target in vitro. Calcium-dependent protein kinases (CDPKs) decode calcium (Ca2+) signals and activate different signaling pathways involved in hormone signaling, plant growth, development, and both abiotic and biotic stress responses. In this study, we describe the potato CDPK/CRK multigene family; bioinformatic analysis allowed us to identify 20 new CDPK isoforms, three CDPK-related kinases (CRKs), and a CDPK-like kinase. Phylogenetic analysis indicated that 26 StCDPKs can be classified into four groups, whose members are predicted to undergo different acylation patterns and exhibited diverse expression levels in different tissues and in response to various stimuli. With the aim of characterizing those members that are particularly involved in plant-pathogen interaction, we focused on StCDPK7. Tissue expression profile revealed that StCDPK7 transcript levels are high in swollen stolons, roots, and mini tubers. Moreover, its expression is induced upon Phytophthora infestans infection in systemic leaves. Transient expression assays showed that StCDPK7 displays a cytosolic/nuclear localization in spite of having a predicted chloroplast transit peptide. The recombinant protein, StCDPK7:6xHis, is an active Ca2+-dependent protein kinase that can phosphorylate phenylalanine ammonia lyase, an enzyme involved in plant defense response. The analysis of the potato CDPK family provides the first step towards the identification of CDPK isoforms involved in biotic stress. StCDPK7 emerges as a relevant player that could be manipulated to deploy disease resistance in potato crops.

Tomato I2 Immune Receptor Can Be Engineered to Confer Partial Resistance to the Oomycete Phytophthora infestans in Addition to the Fungus Fusarium oxysporum.

Plants and animals rely on immune receptors, known as nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins, to defend against invading pathogens and activate immune responses. How NLR receptors respond to pathogens is inadequately understood. We previously reported single-residue mutations that expand the response of the potato immune receptor R3a to AVR3a(EM), a stealthy effector from the late blight oomycete pathogen Phytophthora infestans. I2, another NLR that mediates resistance to the will-causing fungus Fusarium oxysporum f. sp. lycopersici, is the tomato ortholog of R3a. We transferred previously identified R3a mutations to I2 to assess the degree to which the resulting I2 mutants have an altered response. We discovered that wild-type I2 protein responds weakly to AVR3a. One mutant in the N-terminal coiled-coil domain, I2(I141N), appeared sensitized and displayed markedly increased response to AVR3a. Remarkably, I2(I141N) conferred partial resistance to P. infestans. Further, I2(I141N) has an expanded response spectrum to F. oxysporum f. sp. lycopersici effectors compared with the wild-type I2 protein. Our results suggest that synthetic immune receptors can be engineered to confer resistance to phylogenetically divergent pathogens and indicate that knowledge gathered for one NLR could be exploited to improve NLR from other plant species.

Translational fusion and redirection to thylakoid lumen as strategies to enhance accumulation of human papillomavirus E7 antigen in tobacco chloroplasts.

Human papillomavirus (HPV) is the causal agent of cervical cancer, one of the most common causes of death in women worldwide, and its E7 antigen is the major candidate for a therapeutic vaccine. The large scale production of E7 by molecular farming that would lead to the development of a safe and inexpensive vaccine is impaired by its low accumulation level in the plant cell. To enhance antigen production in the plastids, two alternative strategies were carried out: the expression of E7 as a translational fusion to ß-glucuronidase enzyme and redirection of E7 into the thylakoid lumen. The use of the ß-glucuronidase as a partner protein turned out to be a successful strategy, antigen expression levels were enhanced between 30 and 40 times relative to unfused E7. Moreover, best accumulation, albeit at a high metabolic cost that compromised biomass production, was obtained redirecting E7 into the thylakoid lumen by the incorporation of the N-terminal transit peptide, Str. Following this approach lumenal E7 production exceeded the stromal by two orders of magnitude. Our results highlight the relevance of exploring different strategies to improve recombinant protein stability for certain transgenes in order to exploit potential advantages of recombinant protein accumulation in chloroplasts.

Single amino acid mutations in the potato immune receptor R3a expand response to Phytophthora effectors.

Both plants and animals rely on nucleotide-binding domain and leucine-rich repeat-containing (NB-LRR or NLR) proteins to respond to invading pathogens and activate immune responses. How plant NB-LRR proteins respond to pathogens is poorly understood. We undertook a gain-of-function random mutagenesis screen of the potato NB-LRR immune receptor R3a to study how this protein responds to the effector protein AVR3a from the oomycete pathogen Phytophthora infestans. R3a response can be extended to the stealthy AVR3aEM isoform of the effector while retaining recognition of AVR3aKI. Each one of eight single amino acid mutations is sufficient to expand the R3a response to AVR3aEM and other AVR3a variants. These mutations occur across the R3a protein, from the N terminus to different regions of the LRR domain. Further characterization of these R3a mutants revealed that at least one of them was sensitized, exhibiting a stronger response than the wild-type R3a protein to AVR3aKI. Remarkably, the N336Y mutation, near the R3a nucleotide-binding pocket, conferred response to the effector protein PcAVR3a4 from the vegetable pathogen P. capsici. This work contributes to understanding how NB-LRR receptor specificity can be modulated. Together with knowledge of pathogen effector diversity, this strategy can be exploited to develop synthetic immune receptors.

Transformation of Solanum tuberosum plastids allows high expression levels of ß-glucuronidase both in leaves and microtubers developed in vitro.

Plastid genome transformation offers an attractive methodology for transgene expression in plants, but for potato, only expression of gfp transgene (besides the selective gene aadA) has been published. We report here successful expression of ß-glucuronidase in transplastomic Solanum tuberosum (var. Desiree) plants, with accumulation levels for the recombinant protein of up to 41% of total soluble protein in mature leaves. To our knowledge, this is the highest expression level reported for a heterologous protein in S. tuberosum. Accumulation of the recombinant protein in soil-grown minitubers was very low, as described in previous reports. Interestingly, microtubers developed in vitro showed higher accumulation of ß-glucuronidase. As light exposure during their development could be the trigger for this high accumulation, we analyzed the effect of light on ß-glucuronidase accumulation in transplastomic tubers. Exposure to light for 8 days increased ß-glucuronidase accumulation in soil-grown tubers, acting as a light-inducible expression system for recombinant protein accumulation in tuber plastids. In this paper we show that plastid transformation in potato allows the highest recombinant protein accumulation in foliar tissue described so far for this food crop. We also demonstrate that in tubers high accumulation is possible and depends on light exposure. Because tubers have many advantages as protein storage organs, these results could lead to new recombinant protein production schemes based on potato.

Field testing, gene flow assessment and pre-commercial studies on transgenic Solanum tuberosum spp. tuberosum (cv. Spunta) selected for PVY resistance in Argentina.

Solanum tuberosum ssp. tuberosum (cv. Spunta) was transformed with a chimeric transgene containing the Potato virus Y (PVY) coat protein (CP) sequence. Screening for PVY resistance under greenhouse conditions yielded over 100 independent candidate lines. Successive field testing of selected lines allowed the identification of two genetically stable PVY-resistant lines, SY230 and SY233, which were further evaluated in field trials at different potato-producing regions in Argentina. In total, more than 2,000 individuals from each line were tested along a 6-year period. While no or negligible PVY infection was observed in the transgenic lines, infection rates of control plants were consistently high and reached levels of up to 70-80%. Parallel field studies were performed in virus-free environments to assess the agronomical performance of the selected lines. Tubers collected from these assays exhibited agronomical traits and biochemical compositions indistinguishable from those of the non-transformed Spunta cultivar. In addition, an interspecific out-crossing trial to determine the magnitude of possible natural gene flow between transgenic line SY233 and its wild relative Solanum chacoense was performed. This trial yielded negative results, suggesting an extremely low probability for such an event to occur.

Recent developments in effector biology of filamentous plant pathogens.

Filamentous pathogens, such as plant pathogenic fungi and oomycetes, secrete an arsenal of effector molecules that modulate host innate immunity and enable parasitic infection. It is now well accepted that these effectors are key pathogenicity determinants that enable parasitic infection. In this review, we report on the most interesting features of a representative set of filamentous pathogen effectors and highlight recent findings. We also list and describe all the linear motifs reported to date in filamentous pathogen effector proteins. Some of these motifs appear to define domains that mediate translocation inside host cells.

High expression level of a foot and mouth disease virus epitope in tobacco transplastomic plants.

Chloroplast transformation has an extraordinary potential for antigen production in plants because of the capacity to accumulate high levels of recombinant proteins and increased biosafety due to maternal plastid inheritance in most crops. In this article, we evaluate tobacco chloroplasts transformation for the production of a highly immunogenic epitope containing amino acid residues 135-160 of the structural protein VP1 of the foot and mouth disease virus (FMDV). To increase the accumulation levels, the peptide was expressed as a fusion protein with the beta-glucuronidase reporter gene (uidA). The recombinant protein represented the 51% of the total soluble proteins in mature leaves, a level higher than those of the Rubisco large subunit, the most abundant protein in the leaf of a wild-type plant. Despite this high accumulation of heterologous protein, the transplastomic plants and wild-type tobacco were phenotypically indistinguishable. The FMDV epitope expressed in transplastomic plants was immunogenic in mice. These results show that transplastomic tobacco express efficiently the recombinant protein, and we conclude that this technology allows the production of large quantities of immunogenic proteins.

Ten things to know about oomycete effectors.

Long considered intractable organisms by fungal genetic research standards, the oomycetes have recently moved to the centre stage of research on plant-microbe interactions. Recent work on oomycete effector evolution, trafficking and function has led to major conceptual advances in the science of plant pathology. In this review, we provide a historical perspective on oomycete genetic research and summarize the state of the art in effector biology of plant pathogenic oomycetes by describing what we consider to be the 10 most important concepts about oomycete effectors.

Potato virus X coat protein fusion to human papillomavirus 16 E7 oncoprotein enhance antigen stability and accumulation in tobacco chloroplast.

Cervical cancer linked to infection with human papillomavirus (HPV) is the third cause of cancer-related death in women. As the virus cannot be propagated in culture, vaccines have been based on recombinant antigens with inherited high-cost production. In a search of alternative cheap production system, E7 HPV type 16 protein, an attractive candidate for anticancer vaccine development, was engineered to be expressed in tobacco chloroplast. In addition, E7 coding sequence was fused to potato virus X coat protein (CP) to compare expression level. Results show that E7CP transcript accumulation reached lower levels than non-fused E7. However, antigen expression levels were higher for fusion protein indicating that CP stabilizes E7 peptide in the chloroplast stroma. These results support viability of transplastomic plants for antigen production and the relevance of improving recombinant peptide stability for certain transgenes to enhance protein accumulation in this organelle.

Accumulation of hEGF and hEGF-fusion proteins in chloroplast-transformed tobacco plants is higher in the dark than in the light.

Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that heterologous protein accumulation in chloroplasts could be hindered by post-transcriptional mechanisms not yet characterized. Here, we describe the development and characterization of transplastomic tobacco plants transformed with four different transformation vectors for the expression of human epidermal growth factor (hEGF). We showed that, although the corresponding transcript was present in all of the analyzed plants, hEGF could only be detected when fused to the first 186 amino acids of bacterial beta-glucuronidase (GUS). In addition, we observed that the expression levels of recombinant protein increased when plants were placed in the dark or when leaves were incubated in the presence of electron transport inhibitors, such as methyl viologen (MV) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). These results suggest that the mechanism responsible for hEGF instability in chloroplasts is regulated by light.

Insulin-like growth factor-1 receptor regulation in activated human T lymphocytes.

OBJECTIVE: To investigate the kinetics of insulin-like growth factor-1 receptor (IGF-1R) expression in PHA-stimulated T lymphocytes. METHODS: IGF-1R protein and mRNA were detected by flow cytometry and RT-PCR respectively, between 0 and 48 h after cell activation. RESULTS: Few minutes after T lymphocytes were activated, internalization of the IGF-1R from the cell membrane was observed, achieving the lower level between 1 and 6 h and was accompanied by a reduction in its mRNA. This was followed by re-expression of IGF-1R on the cell surface and an increase in IGF-1R mRNA levels in the cytoplasm, reaching levels higher than those recorded initially after 48 h activation. CONCLUSION: This down- and up-regulation suggests that restoration of IGF-1R would be the result of receptor recycling and de novo synthesis and highlights its importance for T lymphocyte proliferation.