Blue-light receptor phototropin 1 suppresses immunity to promote Phytophthora infestans infection.

Blue-light (BL) phototropin receptors (phot1 and phot2) regulate plant growth by activating NPH3/RPT2-like (NRL) family members. Little is known about roles for BL and phots in regulating plant immunity. We showed previously that Phytophthora infestans RXLR effector Pi02860 targets potato (St)NRL1, promoting its ability to enhance susceptibility by facilitating proteasome-mediated degradation of the immune regulator StSWAP70. This raises the question: do BL and phots negatively regulate immunity? We employed coimmunoprecipitation, virus-induced gene silencing, transient overexpression and targeted mutation to investigate contributions of phots to regulating immunity. Whereas transient overexpression of Stphot1 and Stphot2 enhances P. infestans colonization of Nicotiana benthamiana, silencing endogenous Nbphot1 or Nbphot2 reduces infection. Stphot1, but not Stphot2, suppressed the INF1-triggered cell death (ICD) immune response in a BL- and NRL1-dependent manner. Stphot1, when coexpressed with StNRL1, promotes degradation of StSWAP70, whereas Stphot2 does not. Kinase-dead Stphot1 fails to suppress ICD, enhance P. infestans colonization or promote StSWAP70 degradation. Critically, BL enhances P. infestans infection, which probably involves phots but not other BL receptors such as cryptochromes and F-box proteins ZTL1 and FKF1. We demonstrate that Stphot1 and Stphot2 play different roles in promoting susceptibility, and Stphot1 kinase activity is required for BL- and StNRL1-mediated immune suppression.

Evolutionarily distinct resistance proteins detect a pathogen effector through its association with different host targets.

Knowledge of the evolutionary processes which govern pathogen recognition is critical to understanding durable disease resistance. We determined how Phytophthora infestans effector PiAVR2 is recognised by evolutionarily distinct resistance proteins R2 and Rpi-mcq1. We employed yeast two-hybrid, co-immunoprecipitation, virus-induced gene silencing, transient overexpression, and phosphatase activity assays to investigate the contributions of BSL phosphatases to R2- and Rpi-mcq1-mediated hypersensitive response (R2 HR and Rpi-mcq1 HR, respectively). Silencing PiAVR2 target BSL1 compromises R2 HR. Rpi-mcq1 HR is compromised only when BSL2 and BSL3 are silenced. BSL1 overexpression increases R2 HR and compromises Rpi-mcq1. However, overexpression of BSL2 or BSL3 enhances Rpi-mcq1 and compromises R2 HR. Okadaic acid, which inhibits BSL phosphatase activity, suppresses both recognition events. Moreover, expression of a BSL1 phosphatase-dead (PD) mutant suppresses R2 HR, whereas BSL2-PD and BSL3-PD mutants suppress Rpi-mcq1 HR. R2 interacts with BSL1 in the presence of PiAVR2, but not with BSL2 and BSL3, whereas no interactions were detected between Rpi-mcq1 and BSLs. Thus, BSL1 activity and association with R2 determine recognition of PiAVR2 by R2, whereas BSL2 and BSL3 mediate Rpi-mcq1 perception of PiAVR2. R2 and Rpi-mcq1 utilise distinct mechanisms to detect PiAVR2 based on association with different BSLs, highlighting central roles of these effector targets for both disease and disease resistance.

Plant pathogen effector utilizes host susceptibility factor NRL1 to degrade the immune regulator SWAP70.

Plant pathogens deliver effectors into plant cells to suppress immunity. Whereas many effectors inactivate positive immune regulators, other effectors associate with negative regulators of immunity: so-called susceptibility (S) factors. Little is known about how pathogens exploit S factors to suppress immunity. Phytophthora infestans RXLR effector Pi02860 interacts with host protein NRL1, which is an S factor whose activity suppresses INF1-triggered cell death (ICD) and is required for late blight disease. We show that NRL1 interacts in yeast and in planta with a guanine nucleotide exchange factor called SWAP70. SWAP70 associates with endosomes and is a positive regulator of immunity. Virus-induced gene silencing of SWAP70 in Nicotiana benthamiana enhances P. infestans colonization and compromises ICD. In contrast, transient overexpression of SWAP70 reduces P. infestans infection and accelerates ICD. Expression of Pi02860 and NRL1, singly or in combination, results in proteasome-mediated degradation of SWAP70. Degradation of SWAP70 is prevented by silencing NRL1, or by mutation of Pi02860 to abolish its interaction with NRL1. NRL1 is a BTB-domain protein predicted to form the substrate adaptor component of a CULLIN3 ubiquitin E3 ligase. A dimerization-deficient mutant, NRL1NQ, fails to interact with SWAP70 but maintains its interaction with Pi02860. NRL1NQ acts as a dominant-negative mutant, preventing SWAP70 degradation in the presence of effector Pi02860, and reducing P. infestans infection. Critically, Pi02860 enhances the association between NRL1 and SWAP70 to promote proteasome-mediated degradation of the latter and, thus, suppress immunity. Preventing degradation of SWAP70 represents a strategy to combat late blight disease.

AVR2 Targets BSL Family Members, Which Act as Susceptibility Factors to Suppress Host Immunity.

To be successful plant pathogens, microbes use "effector proteins" to manipulate host functions to their benefit. Identifying host targets of effector proteins and characterizing their role in the infection process allow us to better understand plant-pathogen interactions and the plant immune system. Yeast two-hybrid analysis and coimmunoprecipitation were used to demonstrate that the Phytophthora infestans effector AVIRULENCE 2 (PiAVR2) interacts with all three BRI1-SUPPRESSOR1-like (BSL) family members from potato (Solanum tuberosum). Transient expression of BSL1, BSL2, and BSL3 enhanced P. infestans leaf infection. BSL1 and BSL3 suppressed INFESTIN 1 elicitin-triggered cell death, showing that they negatively regulate immunity. Virus-induced gene silencing studies revealed that BSL2 and BSL3 are required for BSL1 stability and show that basal levels of immunity are increased in BSL-silenced plants. Immune suppression by BSL family members is dependent on the brassinosteroid-responsive host transcription factor CIB1/HBI1-like 1. The P. infestans effector PiAVR2 targets all three BSL family members in the crop plant S. tuberosum These phosphatases, known for their role in growth-promoting brassinosteroid signaling, all support P. infestans virulence and thus can be regarded as susceptibility factors in late blight infection.

RXLR Effector AVR2 Up-Regulates a Brassinosteroid-Responsive bHLH Transcription Factor to Suppress Immunity.

An emerging area in plant research focuses on antagonism between regulatory systems governing growth and immunity. Such cross talk represents a point of vulnerability for pathogens to exploit. AVR2, an RXLR effector secreted by the potato blight pathogen Phytophthora infestans, interacts with potato BSL1, a putative phosphatase implicated in growth-promoting brassinosteroid (BR) hormone signaling. Transgenic potato (Solanum tuberosum) plants expressing the effector exhibit transcriptional and phenotypic hallmarks of overactive BR signaling and show enhanced susceptibility to P. infestans Microarray analysis was used to identify a set of BR-responsive marker genes in potato, all of which are constitutively expressed to BR-induced levels in AVR2 transgenic lines. One of these genes was a bHLH transcription factor, designated StCHL1, homologous to AtCIB1 and AtHBI1, which are known to facilitate antagonism between BR and immune responses. Transient expression of either AVR2 or CHL1 enhanced leaf colonization by P. infestans and compromised immune cell death activated by perception of the elicitin Infestin1 (INF1). Knockdown of CHL1 transcript using Virus-Induced Gene Silencing (VIGS) reduced colonization of P. infestans on Nicotiana benthamiana Moreover, the ability of AVR2 to suppress INF1-triggered cell death was attenuated in NbCHL1-silenced plants, indicating that NbCHL1 was important for this effector activity. Thus, AVR2 exploits cross talk between BR signaling and innate immunity in Solanum species, representing a novel, indirect mode of innate immune suppression by a filamentous pathogen effector.

Potato NPH3/RPT2-Like Protein StNRL1, Targeted by a Phytophthora infestans RXLR Effector, Is a Susceptibility Factor.

Plant pathogens deliver effectors to manipulate host processes. We know little about how fungal and oomycete effectors target host proteins to promote susceptibility, yet such knowledge is vital to understand crop disease. We show that either transient expression in Nicotiana benthamiana, or stable transgenic expression in potato (Solanum tuberosum), of the Phytophthora infestans RXLR effector Pi02860 enhances leaf colonization by the pathogen. Expression of Pi02860 also attenuates cell death triggered by the P. infestans microbe-associated molecular pattern INF1, indicating that the effector suppresses pattern-triggered immunity. However, the effector does not attenuate cell death triggered by Cf4/Avr4 coexpression, showing that it does not suppress all cell death activated by cell surface receptors. Pi02860 interacts in yeast two-hybrid assays with potato NPH3/RPT2-LIKE1 (NRL1), a predicted CULLIN3-associated ubiquitin E3 ligase. Interaction of Pi02860 in planta was confirmed by coimmunoprecipitation and bimolecular fluorescence complementation assays. Virus-induced gene silencing of NRL1 in N. benthamiana resulted in reduced P. infestans colonization and accelerated INF1-mediated cell death, indicating that this host protein acts as a negative regulator of immunity. Moreover, whereas NRL1 virus-induced gene silencing had no effect on the ability of the P. infestans effector Avr3a to suppress INF1-mediated cell death, such suppression by Pi02860 was significantly attenuated, indicating that this activity of Pi02860 is mediated by NRL1. Transient overexpression of NRL1 resulted in the suppression of INF1-mediated cell death and enhanced P. infestans leaf colonization, demonstrating that NRL1 acts as a susceptibility factor to promote late blight disease.

PGE(2) induces macrophage IL-10 production and a regulatory-like phenotype via a protein kinase A-SIK-CRTC3 pathway.

The polarization of macrophages into a regulatory-like phenotype and the production of IL-10 plays an important role in the resolution of inflammation. We show in this study that PGE(2), in combination with LPS, is able to promote an anti-inflammatory phenotype in macrophages characterized by high expression of IL-10 and the regulatory markers SPHK1 and LIGHT via a protein kinase A-dependent pathway. Both TLR agonists and PGE(2) promote the phosphorylation of the transcription factor CREB on Ser(133). However, although CREB regulates IL-10 transcription, the mutation of Ser(133) to Ala in the endogenous CREB gene did not prevent the ability of PGE(2) to promote IL-10 transcription. Instead, we demonstrate that protein kinase A regulates the phosphorylation of salt-inducible kinase 2 on Ser(343), inhibiting its ability to phosphorylate CREB-regulated transcription coactivator 3 in cells. This in turn allows CREB-regulated transcription coactivator 3 to translocate to the nucleus where it serves as a coactivator with the transcription factor CREB to induce IL-10 transcription. In line with this, we find that either genetic or pharmacological inhibition of salt-inducible kinases mimics the effect of PGE(2) on IL-10 production.

CREB phosphorylation at Ser133 regulates transcription via distinct mechanisms downstream of cAMP and MAPK signalling.

CREB (cAMP-response-element-binding protein) is an important transcription factor for the activation of a number of immediate early genes. CREB is phosphorylated on Ser133 by PKA (protein kinase A), promoting the recruitment of the co-activator proteins CBP (CREB-binding protein) and p300; this has been proposed to increase the transcription of CREB-dependent genes. CREB is also phosphorylated on Ser133 by MSK1/2 (mitogen- and stress-activated kinase 1/2) in cells in response to the activation of MAPK (mitogen-activated protein kinase) signalling; however, the relevance of this to gene transcription has been controversial. To resolve this problem, we created a mouse with a Ser133 to alanine residue mutation in the endogenous Creb gene. Unlike the total CREB knockout, which is perinatally lethal, these mice were viable, but born at less than the expected Mendelian frequency on a C57Bl/6 background. Using embryonic fibroblasts from the S133A-knockin mice we show in the present study that Ser133 phosphorylation downstream of PKA is required for CBP/p300 recruitment. The requirement of Ser133 phosphorylation for the PKA-mediated induction of CREB-dependent genes was, however, promoter-specific. Furthermore, we show that in cells the phosphorylation of CREB on Ser133 by MSKs does not promote strong recruitment of CBP or p300. Despite this, MSK-mediated CREB phosphorylation is critical for the induction of CREB-dependent genes downstream of MAPK signalling.

Targeted transcriptomic and metabolic profiling reveals temporal bottlenecks in the maize carotenoid pathway that may be addressed by multigene engineering.

Carotenoids are a diverse group of tetraterpenoid pigments found in plants, fungi, bacteria and some animals. They play vital roles in plants and provide important health benefits to mammals, including humans. We previously reported the creation of a diverse population of transgenic maize plants expressing various carotenogenic gene combinations and exhibiting distinct metabolic phenotypes. Here we performed an in-depth targeted mRNA and metabolomic analysis of the pathway to characterize the specific impact of five carotenogenic transgenes and their interactions with 12 endogenous genes in four transgenic lines representing distinct genotypes and phenotypes. We reconstructed the temporal profile of the carotenoid pathway during endosperm development at the mRNA and metabolic levels (for total and individual carotenoids), and investigated the impact of transgene expression on the endogenous pathway. These studies enabled us to investigate the extent of any interactions between the introduced transgenic and native partial carotenoid pathways during maize endosperm development. Importantly, we developed a theoretical model that explains these interactions, and our results suggest genetic intervention points that may allow the maize endosperm carotenoid pathway to be engineered in a more effective and predictable manner.

Characterization of the cellular action of the MSK inhibitor SB-747651A.

MSK1 (mitogen- and stress-activated kinase 1) and MSK2 are nuclear protein kinases that regulate transcription downstream of the ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38α MAPKs (mitogen-activated protein kinases) via the phosphorylation of CREB (cAMP-response-element-binding protein) and histone H3. Previous studies on the function of MSKs have used two inhibitors, H89 and Ro 31-8220, both of which have multiple off-target effects. In the present study, we report the characterization of the in vitro and cellular properties of an improved MSK1 inhibitor, SB-747651A. In vitro, SB-747651A inhibits MSK1 with an IC50 value of 11 nM. Screening of an in vitro panel of 117 protein kinases revealed that, at 1 µM, SB-747651A inhibited four other kinases, PRK2 (double-stranded-RNA-dependent protein kinase 2), RSK1 (ribosomal S6 kinase 1), p70S6K (S6K is S6 kinase) (p70RSK) and ROCK-II (Rho-associated protein kinase 2), with a similar potency to MSK1. In cells, SB-747651A fully inhibited MSK activity at 5-10 µM. SB-747651A was found to inhibit the production of the anti-inflammatory cytokine IL-10 (interleukin-10) in wild-type, but not MSK1/2-knockout, macrophages following LPS (lipopolysaccharide) stimulation. Both SB-747651A and MSK1/2 knockout resulted in elevated pro-inflammatory cytokine production by macrophages in response to LPS. Comparison of the effects of SB-747651A, both in vitro and in cells, demonstrated that SB-747651A exhibited improved selectivity over H89 and Ro 31-8220 and therefore represents a useful tool to study MSK function in cells.

Transgenic rice grains expressing a heterologous ρ-hydroxyphenylpyruvate dioxygenase shift tocopherol synthesis from the γ to the α isoform without increasing absolute tocopherol levels.

We generated transgenic rice plants overexpressing Arabidopsis thaliana ρ-hydroxyphenylpyruvate dioxygenase (HPPD), which catalyzes the first committed step in vitamin E biosynthesis. Transgenic grains accumulated marginally higher levels of total tocochromanols than controls, reflecting a small increase in absolute tocotrienol synthesis (but no change in the relative abundance of the α and γ isoforms). In contrast, there was no change in the absolute tocopherol level, but a significant shift from the γ to the α isoform. These data confirm HPPD is not rate limiting, and that increasing flux through the early pathway reveals downstream bottlenecks that act as metabolic tipping points.

Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways.

Vitamin deficiency affects up to 50% of the world's population, disproportionately impacting on developing countries where populations endure monotonous, cereal-rich diets. Transgenic plants offer an effective way to increase the vitamin content of staple crops, but thus far it has only been possible to enhance individual vitamins. We created elite inbred South African transgenic corn plants in which the levels of 3 vitamins were increased specifically in the endosperm through the simultaneous modification of 3 separate metabolic pathways. The transgenic kernels contained 169-fold the normal amount of beta-carotene, 6-fold the normal amount of ascorbate, and double the normal amount of folate. Levels of engineered vitamins remained stable at least through to the T3 homozygous generation. This achievement, which vastly exceeds any realized thus far by conventional breeding alone, opens the way for the development of nutritionally complete cereals to benefit the world's poorest people.

Synergistic metabolism in hybrid corn indicates bottlenecks in the carotenoid pathway and leads to the accumulation of extraordinary levels of the nutritionally important carotenoid zeaxanthin.

Lutein and zeaxanthin cannot be synthesized de novo in humans, and although lutein is abundant in fruit and vegetables, good dietary sources of zeaxanthin are scarce. Certain corn varieties provide adequate amounts because the ratio of endosperm ß:ε lycopene cyclase activity favours the ß-carotene/zeaxanthin branch of the carotenoid pathway. We previously described a transgenic corn line expressing the early enzymes in the pathway (including lycopene ß-cyclase) and therefore accumulating extraordinary levels of ß-carotene. Here, we demonstrate that introgressing the transgenic mini-pathway into wild-type yellow endosperm varieties gives rise to hybrids in which the ß:ε ratio is altered additively. Where the ß:ε ratio in the genetic background is high, introgression of the mini-pathway allows zeaxanthin production at an unprecedented 56 µg/g dry weight. This result shows that metabolic synergy between endogenous and heterologous pathways can be used to enhance the levels of nutritionally important metabolites.

Simultaneous expression of Arabidopsis ρ-hydroxyphenylpyruvate dioxygenase and MPBQ methyltransferase in transgenic corn kernels triples the tocopherol content.

The quantity and composition of tocopherols (compounds with vitamin E activity) vary widely among different plant species reflecting the expression, activity and substrate specificity of enzymes in the corresponding metabolic pathway. Two Arabidopsis cDNA clones corresponding to ρ-hydroxyphenylpyruvate dioxygenase (HPPD) and 2-methyl-6-phytylplastoquinol methyltransferase (MPBQ MT) were constitutively expressed in corn to further characterize the pathway and increase the kernel tocopherol content. Transgenic kernels contained up to 3 times as much γ-tocopherol as their wild type counterparts whereas other tocopherol isomers remained undetectable. Biofortification by metabolic engineering offers a sustainable alternative to vitamin E supplementation for the improvement of human health.

Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize.

Combinatorial nuclear transformation is a novel method for the rapid production of multiplex-transgenic plants, which we have used to dissect and modify a complex metabolic pathway. To demonstrate the principle, we transferred 5 carotenogenic genes controlled by different endosperm-specific promoters into a white maize variety deficient for endosperm carotenoid synthesis. We recovered a diverse population of transgenic plants expressing different enzyme combinations and showing distinct metabolic phenotypes that allowed us to identify and complement rate-limiting steps in the pathway and to demonstrate competition between beta-carotene hydroxylase and bacterial beta-carotene ketolase for substrates in 4 sequential steps of the extended pathway. Importantly, this process allowed us to generate plants with extraordinary levels of beta-carotene and other carotenoids, including complex mixtures of hydroxycarotenoids and ketocarotenoids. Combinatorial transformation is a versatile approach that could be used to modify any metabolic pathway and pathways controlling other biochemical, physiological, or developmental processes.

Promoter diversity in multigene transformation.

Multigene transformation (MGT) is becoming routine in plant biotechnology as researchers seek to generate more complex and ambitious phenotypes in transgenic plants. Every nuclear transgene requires its own promoter, so when coordinated expression is required, the introduction of multiple genes leads inevitably to two opposing strategies: different promoters may be used for each transgene, or the same promoter may be used over and over again. In the former case, there may be a shortage of different promoters with matching activities, but repetitious promoter use may in some cases have a negative impact on transgene stability and expression. Using illustrative case studies, we discuss promoter deployment strategies in transgenic plants that increase the likelihood of successful and stable multiple transgene expression.

The regulation of carotenoid pigmentation in flowers.

Carotenoids fulfill many processes that are essential for normal growth and development in plants, but they are also responsible for the breathtaking variety of red-to-yellow colors we see in flowers and fruits. Although such visual diversity helps to attract pollinators and encourages herbivores to distribute seeds, humans also benefit from the aesthetic properties of flowers and an entire floriculture industry has developed on the basis that new and attractive varieties can be produced. Over the last decade, much has been learned about the impact of carotenoid metabolism on flower color development and the molecular basis of flower color. A number of different regulatory mechanisms have been described ranging from the transcriptional regulation of genes involved in carotenoid synthesis to the control of carotenoid storage in sink organs. This means we can now explain many of the natural colorful varieties we see around us and also engineer plants to produce flowers with novel and exciting varieties that are not provided by nature.

Cloning and functional characterization of the maize carotenoid isomerase and ß-carotene hydroxylase genes and their regulation during endosperm maturation.

In order to gain further insight into the partly-characterized carotenoid biosynthetic pathway in corn (Zea mays L.), we cloned cDNAs encoding the enzymes carotenoid isomerase (CRTISO) and ß-carotene hydroxylase (BCH) using endosperm mRNA isolated from inbred line B73. For both enzymes, two distinct cDNAs were identified mapping to different chromosomes. The two crtiso cDNAs (Zmcrtiso1 and Zmcrtiso2) mapped to unlinked genes each containing 12 introns, a feature conserved among all crtiso genes studied thus far. ZmCRTISO1 was able to convert tetra-cis prolycopene to all-trans lycopene but could not isomerize the 15-cis double bond of 9,15,9'-tri-cis-ζ-carotene. ZmCRTISO2 is inactivated by a premature termination codon in B73 corn, but importantly the mutation is absent in other corn cultivars and the active enzyme showed the same activity as ZmCRTISO1. The two bch cDNAs (Zmbch1 and Zmbch2) mapped to unlinked genes each coding sequences containing five introns. ZmBCH1 was able to convert ß-carotene into ß-cryptoxanthin and zeaxanthin, but ZmBCH2 was able to form ß-cryptoxanthin alone and had a lower overall activity than ZmBCH1. All four genes were expressed during endosperm development, with mRNA levels rising in line with carotenoid accumulation (especially zeaxanthin and lutein) until 25 DAP. Thereafter, expression declined for three of the genes, with only Zmcrtiso2 mRNA levels maintained by 30 DAP. We discuss the impact of paralogs with different expression profiles and functions on the regulation of carotenoid synthesis in corn.

Metabolic engineering of ketocarotenoid biosynthesis in higher plants.

Ketocarotenoids such as astaxanthin and canthaxanthin have important applications in the nutraceutical, cosmetic, food and feed industries. Astaxanthin is derived from beta-carotene by 3-hydroxylation and 4-ketolation at both ionone end groups. These reactions are catalyzed by beta-carotene hydroxylase and beta-carotene ketolase, respectively. The hydroxylation reaction is widespread in higher plants, but ketolation is restricted to a few bacteria, fungi, and some unicellular green algae. The recent cloning and characterization of beta-carotene ketolase genes in conjunction with the development of effective co-transformation strategies permitting facile co-integration of multiple transgenes in target plants provided essential resources and tools to produce ketocarotenoids in planta by genetic engineering. In this review, we discuss ketocarotenoid biosynthesis in general, and characteristics and functional properties of beta-carotene ketolases in particular. We also describe examples of ketocarotenoid engineering in plants and we conclude by discussing strategies to efficiently convert beta-carotene to astaxanthin in transgenic plants.

Transgenic strategies for the nutritional enhancement of plants.

The nutrients in the human diet ultimately come from plants. However, all our major food crops lack certain essential vitamins and minerals. Although a varied diet provides adequate nutrition, much of the human population, particularly in developing countries, relies on staple crops, such as rice or maize, which does not provide the full complement of essential nutrients. Malnutrition is a significant public health issue in most of the developing world. One way to address this problem is through the enhancement of staple crops to increase their essential nutrient content. Here, we review the current strategies for the biofortification of crops, including mineral fertilization and conventional breeding but focusing on transgenic approaches which offer the most rapid way to develop high-nutrient commercial cultivars.