Xanthomonas natriuretic peptide is recognized by the Arabidopsis natriuretic peptide receptor 1 and through this interaction triggers similar plant responses to its plant counterpart.

Plant natriuretic peptides (PNPs) are hormone peptides that participate in the regulation of ions and water homeostasis in plants. Xanthomonas citri subsp. citri (Xcc) the causal agent of citrus canker disease also possesses a PNP-like peptide (XacPNP). This peptide, similarly to AtPNP-A, the most studied PNP from Arabidopsis thaliana, causes stomatal aperture and enhances photosynthetic efficiency in plant leaves. Thus, the function that has been attributed to XacPNP is to contribute to maintain photosynthetic efficiency and water homeostasis in plant tissue during the infection process, to create favorable conditions for biotrophic pathogens survival. A PNP receptor (AtPNP-R1) for AtPNP-A has been identified and the AtPNP-A activity in regulation of water homeostasis has been observed to depend on the presence of AtPNP-R1. Here, we demonstrated that both AtPNP-A and XacPNP require the presence of AtPNP-R1 to induce plant stomatal aperture. Also, less necrotic tissue was found in infections with pathogens expressing XacPNP and this was dependent on the presence of AtPNP-R1, suggesting that XacPNP interacts with this receptor to exert its function. Finally, we confirmed that AtPNP-A and XacPNP interact with AtPNP-R1 in planta, which support the idea that XacPNP triggers similar plant responses to its plant counterpart.

DOTAP, a lipidic transfection reagent, triggers Arabidopsis plant defense responses.

MAIN CONCLUSION: DOTAP triggers Arabidopsis thaliana immunity and by priming the defense response is able to reduce bacterial pathogen attack. DOTAP is a cationic lipid widely used as a liposomal transfection reagent and it has recently been identified as a strong activator of the innate immune system in animal cells. Plants are sessile organisms and unlike mammals, that have innate and acquired immunity, plants possess only innate immunity. A key feature of plant immunity is the ability to sense potentially dangerous signals, as it is the case for microbe-associated, pathogen-associated or damage-associated molecular patterns and by doing so, trigger an active defense response to cope with the perturbing stimulus. Here, we evaluated the effect of DOTAP in plant basal innate immunity. An initial plant defense response was induced by the cationic lipid DOTAP in the model plant Arabidopsis thaliana, assessed by callose deposition, reactive oxygen species production, and plant cell death. In addition, a proteomic analysis revealed that these responses are mirrored by changes in the plant proteome, such as up-regulation of proteins related to defense responses, including proteins involved in photorespiration, cysteine and oxylipin synthesis, and oxidative stress response; and down-regulation of enzymes related to photosynthesis. Furthermore, DOTAP was able to prime the defense response for later pathogenic challenges as in the case of the virulent bacterial pathogen Pseudomonas syringae pv. tomato. Disease outcome was diminished in DOTAP-pre-treated leaves and bacterial growth was reduced 100 times compared to mock leaves. Therefore, DOTAP may be considered a good candidate as an elicitor for the study of plant immunity.

Bacterial and plant natriuretic peptides improve plant defence responses against pathogens.

Plant natriuretic peptides (PNPs) have been implicated in the regulation of ions and water homeostasis, and their participation in the plant immune response has also been proposed. Xanthomonas citri ssp. citri contains a gene encoding a PNP-like protein (XacPNP) which has no homologues in other bacteria. XacPNP mimics its Arabidopsis thaliana homologue AtPNP-A by modifying host responses to create favourable conditions for pathogen survival. However, the ability of XacPNP to induce plant defence responses has not been investigated. In order to study further the role of XacPNP in vivo, A. thaliana lines over-expressing XacPNP, lines over-expressing AtPNP-A and AtPNP-A-deficient plants were generated. Plants over-expressing XacPNP or AtPNP-A showed larger stomatal aperture and were more resistant to saline or oxidative stress than were PNP-deficient lines. In order to study further the role of PNP in biotic stress responses, A. thaliana leaves were infiltrated with pure recombinant XacPNP, and showed enhanced expression of genes related to the defence response and a higher resistance to pathogen infections. Moreover, AtPNP-A expression increased in A. thaliana on Pseudomonas syringae pv. tomato (Pst) infection. This evidence led us to analyse the responses of the transgenic plants to pathogens. Plants over-expressing XacPNP or AtPNP-A were more resistant to Pst infection than control plants, whereas PNP-deficient plants were more susceptible and showed a stronger hypersensitive response when challenged with non-host bacteria. Therefore, XacPNP, acquired by horizontal gene transfer, is able to mimic PNP functions, even with an increase in plant defence responses.

Xanthomonas citri ssp. citri requires the outer membrane porin OprB for maximal virulence and biofilm formation.

Xanthomonas citri ssp. citri (Xcc) causes canker disease in citrus, and biofilm formation is critical for the disease cycle. OprB (Outer membrane protein B) has been shown previously to be more abundant in Xcc biofilms compared with the planktonic state. In this work, we showed that the loss of OprB in an oprB mutant abolishes bacterial biofilm formation and adherence to the host, and also compromises virulence and efficient epiphytic survival of the bacteria. Moreover, the oprB mutant is impaired in bacterial stress resistance. OprB belongs to a family of carbohydrate transport proteins, and the uptake of glucose is decreased in the mutant strain, indicating that OprB transports glucose. Loss of OprB leads to increased production of xanthan exopolysaccharide, and the carbohydrate intermediates of xanthan biosynthesis are also elevated in the mutant. The xanthan produced by the mutant has a higher viscosity and, unlike wild-type xanthan, completely lacks pyruvylation. Overall, these results suggest that Xcc reprogrammes its carbon metabolism when it senses a shortage of glucose input. The participation of OprB in the process of biofilm formation and virulence, as well as in metabolic changes to redirect the carbon flux, is discussed. Our results demonstrate the importance of environmental nutrient supply and glucose uptake via OprB for Xcc virulence.

Solanum tuberosum StCDPK1 is regulated by miR390 at the posttranscriptional level and phosphorylates the auxin efflux carrier StPIN4 in vitro, a potential downstream target in potato development.

Among many factors that regulate potato tuberization, calcium and calcium-dependent protein kinases (CDPKs) play an important role. CDPK activity increases at the onset of tuber formation with StCDPK1 expression being strongly induced in swollen stolons. However, not much is known about the transcriptional and posttranscriptional regulation of StCDPK1 or its downstream targets in potato development. To elucidate further, we analyzed its expression in different tissues and stages of the life cycle. Histochemical analysis of StCDPK1::GUS (ß-glucuronidase) plants demonstrated that StCDPK1 is strongly associated with the vascular system in stems, roots, during stolon to tuber transition, and in tuber sprouts. In agreement with the observed GUS profile, we found specific cis-acting elements in StCDPK1 promoter. In silico analysis predicted miR390 to be a putative posttranscriptional regulator of StCDPK1. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis showed ubiquitous expression of StCDPK1 in different tissues which correlated well with Western blot data except in leaves. On the contrary, miR390 expression exhibited an inverse pattern in leaves and tuber eyes suggesting a possible regulation of StCDPK1 by miR390. This was further confirmed by Agrobacterium co-infiltration assays. In addition, in vitro assays showed that recombinant StCDPK1-6xHis was able to phosphorylate the hydrophilic loop of the auxin efflux carrier StPIN4. Altogether, these results indicate that StCDPK1 expression is varied in a tissue-specific manner having significant expression in vasculature and in tuber eyes; is regulated by miR390 at posttranscriptional level and suggest that StPIN4 could be one of its downstream targets revealing the overall role of this kinase in potato development.

StCDPK3 Phosphorylates In Vitro Two Transcription Factors Involved in GA and ABA Signaling in Potato: StRSG1 and StABF1.

Calcium-dependent protein kinases, CDPKs, decode calcium (Ca2+) transients and initiate downstream responses in plants. In order to understand how CDPKs affect plant physiology, their specific target proteins must be identified. In tobacco, the bZIP transcription factor Repression of Shoot Growth (NtRSG) that modulates gibberellin (GA) content is a specific target of NtCDPK1. StCDPK3 from potato is homologous (88% identical) to NtCDPK1 even in its N-terminal variable domain. In this work, we observe that NtRSG is also phosphorylated by StCDPK3. The potato RSG family of transcription factors is composed of three members that share similar features. The closest homologue to NtRSG, which was named StRSG1, was amplified and sequenced. qRT-PCR data indicate that StRSG1 is mainly expressed in petioles, stems, lateral buds, and roots. In addition, GA treatment affected StRSG1 expression. StCDPK3 transcripts were detected in leaves, petioles, stolons, roots, and dormant tubers, and transcript levels were modified in response to GA. The recombinant StRSG1-GST protein was produced and tested as a substrate for StCDPK3 and StCDPK1. 6xHisStCDPK3 was able to phosphorylate the potato StRSG1 in a Ca2+-dependent way, while 6xHisStCDPK1 could not. StCDPK3 also interacts and phosphorylates the transcription factor StABF1 (ABRE binding factor 1) involved in ABA signaling, as shown by EMSA and phosphorylation assays. StABF1 transcripts were mainly detected in roots, stems, and stolons. Our data suggest that StCDPK3 could be involved in the cross-talk between ABA and GA signaling at the onset of tuber development.

Transcript profiling reveals that cysteine protease inhibitors are up-regulated in tuber sprouts after extended darkness.

Potato (Solanum tuberosum L.) tubers are an excellent staple food due to its high nutritional value. When the tuber reaches physiological competence, sprouting proceeds accompanied by changes at mRNA and protein levels. Potato tubers become a source of carbon and energy until sprouts are capable of independent growth. Transcript profiling of sprouts grown under continuous light or dark conditions was performed using the TIGR 10K EST Solanaceae microarray. The profiles analyzed show a core of highly expressed transcripts that are associated to the reactivation of growth. Under light conditions, the photosynthetic machinery was fully activated; the highest up-regulation was observed for the Rubisco activase (RCA), the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the Photosystem II 22 kDa protein (CP22) genes, among others. On the other hand, sprouts exposed to continuous darkness elongate longer, and after extended darkness, synthesis of chloroplast components was repressed, the expression of proteases was reduced while genes encoding cysteine protease inhibitors (CPIs) and metallocarboxypeptidase inhibitors (MPIs) were strongly induced. Northern blot and RT-PCR analysis confirmed that MPI levels correlated with the length of the dark period; however, CPI expression was strong only after longer periods of darkness, suggesting a feedback loop (regulation mechanism) in response to dark-induced senescence. Prevention of cysteine protease activity in etiolated sprouts exposed to extended darkness could delay senescence until they emerge to light.

An Overview of Proteomics Tools for Understanding Plant Defense Against Pathogens.

Plant diseases are responsible for important losses in crops and cause serious impacts in agricultural production. In the last years, proteomics has been used to examine plant defense responses against pathogens. Such studies may be pioneer in the generation of crops with enhanced resistance. In this review, we focus on proteomics advances in the understanding of host and non-host resistance against pathogens.

The BEL1-like family of transcription factors in potato.

BEL1-type proteins are ubiquitous plant transcription factors in the three-amino-acid-loop-extension superfamily. They interact with KNOTTED1-like proteins, and function as heterodimers in both floral and vegetative development. Using the yeast two-hybrid system with POTATO HOMEOBOX1 (POTH1) as the bait, seven BEL1-type proteins were originally identified. One of these genes, designated StBEL5, has transcripts that move long distances in the plant and enhance tuberization and root growth. Using the potato genome database, 13 active BEL1-like genes were identified that contain the conserved homeobox domain and the BELL domain, both of which are essential for the function of BEL1-type proteins. Phylogenetic analysis of the StBEL family demonstrated a degree of orthology with the 13 BEL1-like genes of Arabidopsis. A profile of the gene structure of the family revealed conservation of the length and splicing patterns of internal exons that encode key functional domains. Yeast two-hybrid experiments with KNOTTED1-like proteins and the new StBELs confirmed the interactive network between these two families. Analyses of RNA abundance patterns clearly showed that three StBEL genes, BEL5, -11, and -29, make up approximately two-thirds of the total transcript values for the entire family. Among the 10 organs evaluated here, these three genes exhibited the 12 greatest transcript abundance values. Using a phloem-transport induction system and gel-shift assays, transcriptional cross-regulation within the StBEL family was confirmed. Making use of the potato genome and current experimental data, a comprehensive profile of the StBEL family is presented in this study.

The novel Solanum tuberosum calcium dependent protein kinase, StCDPK3, is expressed in actively growing organs.

Calcium-dependent protein kinases (CDPKs) are key components of calcium regulated signaling cascades in plants. In this work, isoform StCDPK3 from Solanum tuberosum was studied and fully described. StCDPK3 encodes a 63 kDa protein with an N-terminal variable domain (NTV), rich in prolines and glutamines, which presents myristoylation and palmitoylation consensus sites and a PEST sequence indicative of rapid protein degradation. StCDPK3 gene (circa 11 kb) is localized in chromosome 3, shares the eight exons and seven introns structure with other isoforms from subgroup IIa and contains an additional intron in the 5'UTR region. StCDPK3 expression is ubiquitous being transcripts more abundant in early elongating stolons (ES), leaves and roots, however isoform specific antibodies only detected the protein in leaf particulate extracts. The recombinant 6xHis-StCDPK3 is an active kinase that differs in its kinetic parameters and calcium requirements from StCDPK1 and 2 isoforms. In vitro, StCDPK3 undergoes autophosphorylation regardless of the addition of calcium. The StCDPK3 promoter region (circa 1,800 bp) was subcloned by genome walking and fused to GUS. Light and ABRE responsive elements were identified in the promoter region as well as elements associated to expression in roots. StCDPK3 expression was enhanced by ABA while GA decreased it. Potato transgenic lines harboring StCDPK3 promoter∷GUS construct were generated by Agrobacterium tumefaciens mediated plant transformation. Promoter activity was detected in leaves, root tips and branching points, early ES, tuber eyes and developing sprouts indicating that StCDPK3 is expressed in actively growing organs.

Characterization of StABF1, a stress-responsive bZIP transcription factor from Solanum tuberosum L. that is phosphorylated by StCDPK2 in vitro.

ABF/AREB bZIP transcription factors mediate plant abiotic stress responses by regulating the expression of stress-related genes. These proteins bind to the abscisic acid (ABA)-responsive element (ABRE), which is the major cis-acting regulatory sequence in ABA-dependent gene expression. In an effort to understand the molecular mechanisms of abiotic stress resistance in cultivated potato (Solanum tuberosum L.), we have cloned and characterized an ABF/AREB-like transcription factor from potato, named StABF1. The predicted protein shares 45-57% identity with A. thaliana ABFs proteins and 96% identity with the S. lycopersicum SlAREB1 and presents all of the distinctive features of ABF/AREB transcription factors. Furthermore, StABF1 is able to bind to the ABRE in vitro. StABF1 gene is induced in response to ABA, drought, salt stress and cold, suggesting that it might be a key regulator of ABA-dependent stress signaling pathways in cultivated potato. StABF1 is phosphorylated in response to ABA and salt stress in a calcium-dependent manner, and we have identified a potato CDPK isoform (StCDPK2) that phosphorylates StABF1 in vitro. Interestingly, StABF1 expression is increased during tuber development and by tuber-inducing conditions (high sucrose/nitrogen ratio) in leaves. We also found that StABF1 calcium-dependent phosphorylation is stimulated by tuber-inducing conditions and inhibited by gibberellic acid, which inhibits tuberization.

StCDPK2 expression and activity reveal a highly responsive potato calcium-dependent protein kinase involved in light signalling.

Calcium-dependent protein kinases (CDPKs) are essential calcium sensors. In this work, we have studied StCDPK2 isoform from potato both at gene and protein level. StCdpk2 genomic sequence contains eight exons and seven introns, as was observed for StCdpk1. There is one copy of the gene per genome located in chromosome 7. StCDPK2 encodes an active CDPK of 515 aminoacids, with an apparent MW of 57 kDa, which presents myristoylation and palmitoylation consensus in its N-terminus. StCDPK2 is highly expressed in leaves and green sprouts; enhanced expression was detected under light treatment, which corresponds well with light responsive cis-acting elements found in its promoter sequence. Antibodies against the recombinant StCDPK2::6xHis protein detected this isoform in soluble and particulate fractions from leaves. StCDPK2 autophosphorylation and kinase activity are both calcium dependent reaching half maximal activation at 0.6 µM calcium. The active kinase is autophosphorylated on serine and tyrosine residues and its activity is negatively modulated by phosphatidic acid (PA). Our results reveal StCDPK2 as a signalling element involved in plant growth and development and show that its activity is tightly regulated.

Genomic and functional characterization of StCDPK1.

StCDPK1 is a calcium dependent protein kinase expressed in tuberizing potato stolons and in sprouting tubers. StCDPK1 genomic sequence contains eight exons and seven introns, the gene structure is similar to Arabidopsis, rice and wheat CDPKs belonging to subgroup IIa. There is one copy of the gene per genome and it is located in the distal portion of chromosome 12. Western blot and immunolocalization assays (using confocal and transmission electron microscopy) performed with a specific antibody against StCDPK1 indicate that this kinase is mainly located in the plasma membrane of swelling stolons and sprouting tubers. Sucrose (4-8%) increased StCDPK1 protein content in non-induced stolons, however the amount detected in swelling stolons was higher. Transgenic lines with reduced expression of StCDPK1 (beta 7) did not differ from controls when cultured under multiplication conditions, but when grown under tuber inducing conditions some significant differences were observed: the beta 7 line tuberized earlier than controls without the addition of CCC (GA inhibitor), developed more tubers than wild type plants in the presence of hormones that promote tuberization in potato (ABA and BAP) and was more insensitive to GA action (stolons were significantly shorter than those of control plants). StCDPK1 expression was induced by GA, ABA and BAP. Our results suggest that StCDPK1 plays a role in GA-signalling and that this kinase could be a converging point for the inhibitory and promoting signals that influence the onset of potato tuberization.