Oral Immunization With a Plant HSP90-SAG1 Fusion Protein Produced in Tobacco Elicits Strong Immune Responses and Reduces Cyst Number and Clinical Signs of Toxoplasmosis in Mice.

Plant 90kDa heat shock protein (HSP90) is a potent adjuvant that increases both humoral and cellular immune responses to diverse proteins and peptides. In this study, we explored whether Arabidopsis thaliana HSP90 (AtHsp81.2) can improve the immune effects of a Toxoplasma gondii surface antigen 1 (SAG1). We designed two constructs containing the sequence of mature antigen (SAG1m), from aa77 to aa322, and B- and T-cell antigenic epitope-containing SAG1HC, from aa221 to aa319 fused to AtHsp81.2 sequence. When comparing the transient expression in Nicotiana tabacum X-27-8 leaves, which overexpress the suppressor helper component protease HC-Pro-tobacco etch virus (TEV), to that in N. benthamiana leaves, co-agroinfiltrated with the suppressor p19, optimal conditions included 6-week-old N. benthamiana plants, 7-day time to harvest, Agrobacterium tumefaciens cultures with an OD600nm of 0.6 for binary vectors and LED lights. While AtHsp81.2-SAG1m fusion protein was undetectable by Western blot in any of the evaluated conditions, AtHsp81.2-SAG1HC was expressed as intact fusion protein, yielding up to 90µg/g of fresh weight. Besides, the AtHsp81.2-SAG1HC mRNA was strongly expressed compared to the endogenous Nicotiana tabacum elongation factor-alpha (NtEFα) gene, whereas the AtHsp81.2-SAG1m mRNA was almost undetectable. Finally, mice were orally immunized with AtHsp81.2-SAG1HC-infiltrated fresh leaves (plAtHsp81.2-SAG1HC group), recombinant AtHsp81.2-SAG1HC purified from infiltrated leaves (rAtHsp81.2-SAG1HC group), non-infiltrated fresh leaves (control group), or phosphate-buffered saline (PBS group). Serum samples from plAtHsp81.2-SAG1HC-immunized mice had significantly higher levels of IgGt, IgG2a, and IgG2b anti-SAG1HC antibodies than serum from rAtHsp81.2-SAG1HC, control, and PBS groups. The number of cysts per brain in the plAtHsp81.2-SAG1HC-immunized mice was significantly reduced, and the parasite load in brain tissue was also lower in this group compared with the remaining groups. In an immunoblot assay, plant-expressed AtHsp81.2-SAG1HC was shown to react with antibodies present in sera from T. gondii-infected people. Therefore, the plant expression of a T. gondii antigen fused to the non-pathogenic adjuvant and carrier plant HSP90 as formulations against T. gondii can improve the vaccine efficacy, and plant extract can be directly used for vaccination without the need to purify the protein, making this platform a suitable and powerful biotechnological system for immunogenic antigen expression against toxoplasmosis.

Barley plants carrying the altered function Sln1d allele display modified responses to low phosphorus supply: implications for phosphorus utilisation efficiency.

The module GA-GID1-DELLA (Gibberellin-Gibberellin Receptor-DELLA proteins) provides a point for the integration of signals potentially relevant in determining nutrient utilisation and acquisition efficiencies. In this study, we explored the role of components of this module during the acclimation of barley plants (Hordeum vulgare L.) to different phosphorus (P) supplies by using two related genotypes, harbouring either the WT or the Sln1d alleles of the DELLA-coding gene Sln1. Dwarf Sln1d plants exhibited reduced shoot P utilisation efficiency (PUtE) and better performance at low levels of P supply. The superior PUtE displayed by WT plants disappeared when corrected by internal P concentration, indicating that multiple analyses are necessary to fully understand the meaning of PUtE estimates. Over a wide range of external supplies of P, Sln1d plants displayed enhanced P concentration, which was associated with low relative growth rate, high biomass partitioning to roots and high P-uptake-rate, thus suggesting that the effect of the Sln1d allele on P dynamics is not simply a consequence of slow growth habit. An enhanced P concentration was also found in a mutant with defective GAs-synthesis. Our results suggest that components of the GA-GID1-DELLAs module contribute to set the acclimation response of barley plants to low P supply through both P-dynamics dependent and P-dynamics independent mechanisms.

Exposure to a natural nitric oxide donor negatively affects the potential influx of rubidium in potassium-starved Arabidopsis plants.

Nitric oxide (NO) and potassium (K+) exert a profound influence on the acclimation of plants to multiple stress conditions. A recent report indicated that exogenous addition of an NO donor causes, under conditions of adequate K+ supply, a detrimental effect on K+ status. It remains unknown whether an exogenous NO source could negatively affect the potential capture of this element when plants are faced with a K+ shortage. In this work we offer evidence that, under conditions of K+-deprivation, the addition of the naturally occurring NO donor, S-nitrosoglutathione (GSNO), diminishes the potential inward transport of the K+-analogue rubidium (Rb+) from diluted Rb+ concentrations in Arabidopsis thaliana. Studies with the akt1-2 mutant, lacking the AKT1 inward-rectifier K+-channel involved in K+-uptake, unveiled that the effect of GSNO on Rb+-influx involves a non-AKT1 component. In addition, exposure to the NO-donor led to down-regulation of transcripts coding for the AtHAK5 K+-transporter, a major component of the K+-transport machinery in K+-deprived plants. Moreover, studies with the hak5 mutant showed that GSNO could either stimulate Rb+-uptake or does not lead to a significant effect on Rb+-uptake relative to -K+ and to -K+ in the presence of decayed GSNO, respectively, thus indicating that the presence of AtHAK5 is required for GSNO exerting an inhibitory effect.

A survey of sequences of KT-HAK-KUP transporters in green algae and basal land plants.

In this data article, information is provided on sequences of KT-HAK-KUP transporters from green algae and basal land plants. A data set is offered containing sequences corresponding to the chlorophyte algae Chlamydomonas eustigma, Gonium pectorale and Coccomyxa subellipsoidea, the charophyte algae Coleochaete orbicularis and Klebsormidium flaccidum, the bryophyte Sphagnum fallax, the marchantophyte Marchantia polymorpha and the gymnosperm Pinus taeda, which have been not formerly analyzed. In addition, an analysis of similarity scores among representatives of the clusters recognized in photosynthetic green organisms (namely, chlorophyte algae, charophyte algae, basal embryophytes and higher embryophytes) is performed as well as an analysis of membrane topology for them.

KT-HAK-KUP transporters in major terrestrial photosynthetic organisms: A twenty years tale.

Since their discovery, twenty years ago, KT-HAK-KUP transporters have become a keystone to understand how alkali cation fluxes are controlled in major land-dwelling photosynthetic organisms. In this review we focus on their discovery, phylogeny, and functions, as well as the regulation of its canonical member, AtHAK5. We also address issues related to structure-function studies, and the technological possibilities opened up by recent findings. Available evidence suggests that this family of transporters underwent an early divergence into major groups following the conquest of land by embryophytes. KT-HAK-KUPs are necessary to accomplish several major developmental and growth processes, as well as to ensure plant responses to environmental injuries. Although the primary function of these transporters is to mediate potassium (K+) fluxes, some of them can also mediate sodium (Na+) and cesium (Cs+) transport, and contribute to maintenance of K+ (and Na+) homeostasis in different plant tissues. In addition, there is evidence for a role of some members of this family in auxin movement and in adenylate cyclase activity. Recent research, focusing on the regulation of the canonical member of this family, AtHAK5, revealed the existence of a complex network that involves transcriptional and post-transcriptional phenomena which control the enhancement of AtHAK5-mediated K+ uptake when Arabidopsis thaliana plants are faced with low K+ supply. In spite of the formidable advances made since their discovery, important subjects remain to be elucidated to gain a more complete knowledge of the roles and regulation of KT-HAK-KUPs, as well as to improve their use for innovative procedures in crop breeding.

How DELLAs contribute to control potassium uptake under conditions of potassium scarcity? Hypotheses and uncertainties.

Maintenance of the inward transport of potassium (K) by roots is a critical step to ensure K-nutrition for all plant tissues. When plants are grown at low external K concentrations a strong enhancement of the activity of the AtHAK5 transporter takes place. In a recent work, we observed that the gai-1 mutant of Arabidopsis thaliana, which bears an altered function version of a DELLA regulatory protein, displays reduced accumulation of AtHAK5 transcripts and reduced uptake of Rubidium, an analog for K. In this Addendum we discuss some hypotheses and uncertainties regarding how DELLAs could contribute to the control of K uptake under those conditions. We advance the idea that, following K-restriction, there is a zone and tissue specific regulation of DELLAs by gibberellins through a pathway that likely involves ethylene. According to this model in the epidermis of non-apical zones, DELLAs repress transcription factors that promote AtHAK5 accumulation.

Internal efficiency of nutrient utilization: what is it and how to measure it during vegetative plant growth?

Efficient use of the resources required by plants to sustain crop production is considered an important objective in agriculture. In this context, the idea of developing crops with an enhanced ability to utilize mineral nutrients already taken up by roots has been proposed. In recent years powerful tools that allow the association of phenotypic variation with high-resolution genetic maps of crop plants have also emerged. To take advantage of these tools, accurate methods are needed to estimate the internal efficiency of nutrient utilization (ENU) at the whole-plant level, which requires using suitable conceptual and experimental approaches. Here we highlight some inconsistencies in the definitions of ENU commonly used for ENU 'phenotyping' at the vegetative stage and suggest that it would be convenient to adopt a dynamic definition. The idea that ENU should provide information about the relationship between carbon and mineral nutrient economies mainly during the period under which growth is actually affected by low internal nutrient concentration is here advocated as a guide for the selection of adequate operational ENU formulae for the vegetative stage. The desirability of using experimental approaches that allow removal of the influence of nutrient acquisition efficiency on ENU estimations is highlighted. It is proposed that the use of simulation models could help refine the conclusions obtained through these experimental procedures. Some potential limitations in breeding for high ENU are also considered.