It takes two to tango : molecular links between plant immunity and brassinosteroid signalling

In response to the invasion of microorganisms, plants actively balance their resources for growth and defence, thus ensuring their survival. The regulatory mechanisms underlying plant immunity and growth operate through complex networks, in which the brassinosteroid phytohormone is one of the central players. In the past decades, a growing number of studies have revealed a multi-layered crosstalk between brassinosteroid-mediated growth and plant immunity. In this Review, by means of the tango metaphor, we immerse ourselves into the intimate relationship between brassinosteroid and plant immune signalling pathways that is tailored by the lifestyle of the pathogen and modulated by other phytohormones. The plasma membrane is the unique stage where brassinosteroid and immune signals are dynamically integrated and where compartmentalization into nanodomains that host distinct protein consortia is crucial for the dance. Shared downstream signalling components and transcription factors relay the tango play to the nucleus to activate the plant defence response and other phytohormonal signalling pathways for the finale. Understanding how brassinosteroid and immune signalling pathways are integrated in plants will help develop strategies to minimize the growth-defence trade-off, a key challenge for crop improvement

Global analysis of the sugarcane microtranscriptome reveals a unique composition of small RNAs associated with axillary bud outgrowth

Axillary bud outgrowth determines shoot architecture and is under the control of endogenous hormones and a fine-tuned gene-expression network, which probably includes small RNAs (sRNAs). Although it is well known that sRNAs act broadly in plant development, our understanding about their roles in vegetative bud outgrowth remains limited. Moreover, the expression profiles of microRNAs (miRNAs) and their targets within axillary buds are largely unknown. Here, we employed sRNA next-generation sequencing as well as computational and gene-expression analysis to identify and quantify sRNAs and their targets in vegetative axillary buds of the biofuel crop sugarcane (Saccharum spp.). Computational analysis allowed the identification of 26 conserved miRNA families and two putative novel miRNAs, as well as a number of trans-acting small interfering RNAs. sRNAs associated with transposable elements and protein-encoding genes were similarly represented in both inactive and developing bud libraries. Conversely, sequencing and quantitative reverse transcription-PCR results revealed that specific miRNAs were differentially expressed in developing buds, and some correlated negatively with the expression of their targets at specific stages of axillary bud development. For instance, the expression patterns of miR159 and its target GAMYB suggested that they may play roles in regulating abscisic acid-signalling pathways during sugarcane bud outgrowth. Our work reveals, for the first time, differences in the composition and expression profiles of diverse sRNAs and targets between inactive and developing vegetative buds that, together with the endogenous balance of specific hormones, may be important in regulating axillary bud outgrowth

Identification and expression analysis of microRNAs and targets in the biofuel crop sugarcane

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) are small regulatory RNAs, some of which are conserved in diverse plant genomes. Therefore, computational identification and further experimental validation of miRNAs from non-model organisms is both feasible and instrumental for addressing miRNA-based gene regulation and evolution. Sugarcane (<it>Saccharum spp</it>.) is an important biofuel crop with publicly available expressed sequence tag and genomic survey sequence databases, but little is known about miRNAs and their targets in this highly polyploid species.</p> <p>Results</p> <p>In this study, we have computationally identified 19 distinct sugarcane miRNA precursors, of which several are highly similar with their sorghum homologs at both nucleotide and secondary structure levels. The accumulation pattern of mature miRNAs varies in organs/tissues from the commercial sugarcane hybrid as well as in its corresponding founder species <it>S. officinarum </it>and <it>S. spontaneum</it>. Using sugarcane <it>MIR827 </it>as a query, we found a novel <it>MIR827 </it>precursor in the sorghum genome. Based on our computational tool, a total of 46 potential targets were identified for the 19 sugarcane miRNAs. Several targets for highly conserved miRNAs are transcription factors that play important roles in plant development. Conversely, target genes of lineage-specific miRNAs seem to play roles in diverse physiological processes, such as <it>SsCBP1</it>. <it>SsCBP1 </it>was experimentally confirmed to be a target for the monocot-specific miR528. Our findings support the notion that the regulation of <it>SsCBP1 </it>by miR528 is shared at least within graminaceous monocots, and this miRNA-based post-transcriptional regulation evolved exclusively within the monocots lineage after the divergence from eudicots.</p> <p>Conclusions</p> <p>Using publicly available nucleotide databases, 19 sugarcane miRNA precursors and one new sorghum miRNA precursor were identified and classified into 14 families. Comparative analyses between sugarcane and sorghum suggest that these two species retain homologous miRNAs and targets in their genomes. Such conservation may help to clarify specific aspects of miRNA regulation and evolution in the polyploid sugarcane. Finally, our dataset provides a framework for future studies on sugarcane RNAi-dependent regulatory mechanisms.</p

Functional and evolutionary analyses of the miR156 and miR529 families in land plants

Abstract\ud \ud Background\ud MicroRNAs (miRNAs) are important regulatory elements of gene expression. Similarly to coding genes, miRNA genes follow a birth and death pattern of evolution likely reflecting functional relevance and divergence. For instance, miRNA529 is evolutionarily related to miRNA156 (a highly conserved miRNA in land plants), but it is lost in Arabidopsis thaliana. Interestingly, both miRNAs target sequences overlap in some members of the SQUAMOSA promoter-binding protein like (SPL) family, raising important questions regarding the diversification of the miR156/miR529-associated regulatory network in land plants.\ud \ud \ud Results\ud In this study, through phylogenic reconstruction of miR156/529 target sequences from several taxonomic groups, we have found that specific eudicot SPLs, despite miRNA529 loss, retained the corresponding target site. Detailed molecular evolutionary analyses of miR156/miR529-target sequence showed that loss of miR529 in core eudicots, such as Arabidopsis, is correlated with a more relaxed selection of the miRNA529 specific target element, while miRNA156-specific target sequence is under stronger selection, indicating that these two target sites might be under distinct evolutionary constraints. Importantly, over-expression in Arabidopsis of MIR529 precursor from a monocot, but not from a basal eudicot, demonstrates specific miR529 regulation of AtSPL9 and AtSPL15 genes, which contain conserved responsive elements for both miR156 and miR529.\ud \ud \ud Conclusions\ud Our results suggest loss of functionality of MIR529 genes in the evolutionary history of eudicots and show that the miR529-responsive element present in some eudicot SPLs is still functional. Our data support the notion that particular miRNA156 family members might have compensated for the loss of miR529 regulation in eudicot species, which concomitantly may have favored diversification of eudicot SPLs.We thank Dr. Scott Poethig for 35S::AtMIR156a seeds; Dr. Peter Huijser for\ud spl9;spl15 seeds; Dr. Renato Vicentini for initial bioinformatic analyses and\ud helpful discussions; and Dr. Luiz Del Bem for initial phylogenetic analyses.\ud This work was supported by the State of Sao Paulo Research Foundation,\ud FAPESP, Brazil (grants no. 07/58289-5 and 12/51146-2). EGOM was a recipient\ud of a fellowship from Coordination for the Improvement of Higher Education\ud Personnel (CAPES, Brazil). GFFS (from Centro de Energia Nuclear na\ud Agricultura –CENA/USP) and EMS were recipients of a fellowship from the\ud State of Sao Paulo Research Foundation, FAPESP, Brazil

Protein 3D Structure Computed from Evolutionary Sequence Variation

The evolutionary trajectory of a protein through sequence space is constrained by its function. Collections of sequence homologs record the outcomes of millions of evolutionary experiments in which the protein evolves according to these constraints. Deciphering the evolutionary record held in these sequences and exploiting it for predictive and engineering purposes presents a formidable challenge. The potential benefit of solving this challenge is amplified by the advent of inexpensive high-throughput genomic sequencing

Dinâmica subcelular do peptídeo endógeno AtPep1 e seus receptores em Arabidopsis: implicações na imunidade de plantas

This work investigated the subcellular dynamics of the plant elicitor peptide AtPep1 and its interplay with plant defense responses. First, an introduction of the plant innate immunity system is provided with emphasis on pattern trigger immunity (PTI), which is based on the recognition of \"non-self\" and \"self\" elicitor molecules by surface-localized patternrecognition receptors (PRRs). Then, the Arabidopsis endogenous peptides that act as selfelicitor molecules are presented, with details on AtPep1 and its PEPR receptors. Plant endomembrane trafficking is described, encompassing endocytic pathways, clathrin mediated endocytosis (CME) and receptor-mediated endocytosis (RME). In the next chapter, we explored strategies for the in vivo study of the subcellular behavior of AtPep1; to this end, we fused the precursor protein of AtPep1 (PROPEP1) to GFP and assessed its localization. We found that PROPEP1 was associated with the tonoplast and accumulated in the vacuole, suggesting that this organelle could work as the station where PROPEP1 is stored and later released, only in a danger situation, hence initiating AtPep1. Moreover, we generated AtPep1 versions labeled with fluorescent dyes and demonstrated that this peptide could be fluorescently tagged without loss of its biological activity. In chapter 3, we combined classical and chemical genetics with life imaging to study the behavior of a bioactive fluorescently labeled AtPep1 in the Arabidopsis root meristem. We discovered that the labeled AtPep1 was able to bind the plasma membrane very quickly in a receptor-dependent manner. Subsequently, the PEPR-AtPep1 complex was internalized via CME and transported to the lytic vacuole, passing through early and late endosomal compartments. Impairment of CME compromised the AtPep1 responses. Our findings provide for the first time an in vivo visualization of a signaling peptide in plant cells, thus giving insights into its intracellular fate and dynamics. The role of the coregulatory receptor BRI1-associated kinase 1 (BAK1) in AtPep1-responses was also investigated (chapter 4). Our results confirmed that BAK1 interacts with PEPRs in a ligand-dependent manner and indicate that BAK1 modulates AtPep1 signaling and endocytosis, but that, when absent, it might be replaced by homologous SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) proteins that could have additional functions during the AtPep1 signaling. Furthermore, phosphorylation events after the formation of PEPR-BAK1 complexes seem to dictate the molecular bases of AtPep1 internalization and signaling. Finally, we discussed our findings in a more general perspective, highlighting the important findings for the plant endomembrane trafficking field, the potential use of fluorescently labeled ligands as a tool to study ligand-receptors pairs, the availability of AtPep1-PEPRs as an excellent model to study endocytosis and its interplay with signaling, and the future challenges in the field.Neste trabalho, foi investigada a dinâmica subcelular do peptídeo elicitor de planta AtPep1 e suas implicações nas respostas de defesa. Primeiramente, é fornecida uma introdução do sistema imune inato de plantas com ênfase na imunidade ativada por moléculas elicitoras derivadas de organismos invasores ou da mesma planta, após seu reconhecimento por receptores localizados na membrana plasmática (PTI responses). Peptídeos endógenos que têm sido reportados em Arabidopsis como ativadores de PTI são descritos, dando especial destaque para o peptídeo AtPep1 e seus receptores PEPRs. O tráfego de endomembranas em plantas é introduzido, abrangendo as vias de internalização, endocitose mediada por proteínas clathrinas (CME) e endocitose mediada por receptor (RME). No capítulo seguinte, foram avaliadas estratégias para o estudo in vivo da dinâmica subcelular do AtPep1. Para isso a proteína precursora do AtPep1 (PROPEP1) foi fusionada a GFP e sua localização visualizada, encontrando que PROPEP1 é associado com o tonoplasto e acumula dentro do vacúolo, fato que sugere uma função de armazenamento do PROPEP1 para esta organela, desde onde é liberado em caso de uma situação de perigo dando origem ao AtPep1. Adicionalmente, foram produzidas versões biologicamente ativas do AtPep1 marcado com fluróforos. No capítulo três foram combinados genética clássica e genética química com visualizações in vivo para estudar o comportamento de um AtPep1 bioativo e marcado fluorescentemente na células meristemática da ponta da raiz de Arabidopsis, sendo encontrado que AtPep1 se liga rapidamente na membrana plasmática numa forma dependente de receptor. Em seguida, o complexo AtPep1-PEPR foi internalizado via CME e transportado para o vacúolo, passando através do endossomo primário e secundário. Quando o funcionamento da CME foi comprometido, as respostas ao AtPep1 também foram afetadas. Estes resultados fornecem a primeira visualização in vivo de um peptídeo de sinalização em plantas, mostrando sua dinâmica e destino intracelular. O papel regulatório durante as respostas induzidas pelo AtPep1 do co-receptor BRI1-associated kinase 1 (BAK1) foram investigadas (Capítulo quatro). Nossos resultados confirmaram que BAK1 interage com PEPRs numa forma dependente do ligante e indicam que BAK1 modula sinalização e endocitose do AtPep1, no entanto quando ausente, BAK1 pode ser substituído por seus homólogos SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE os quais poderiam ter funções adicionais durante as repostas induzidas pelo AtPep1. Eventos de fosforilação após a formação do complexo PEPR-BAK1 parecem ditar as bases moleculares da internalização e sinalização do AtPep1. Finalmente, são discutidos os resultados encontrados nesta pesquisa numa perspectiva geral, destacando a relevância destas descobertas na área de pesquisa em que estão inseridos, o potencial que representa o uso de ligantes marcados fluorescentemente como ferramenta para o estudo de complexos entre ligante-receptor, a disponibilidade do sistema AtPep1-PEPRs como modelo de estudo da endocitose em plantas e sua relação com sinalização, e os futuros desafios na área

Analysis of the role(s) of the miRNA156/SPL pathway on branching/tillering and molecular characterization of sugarcane lateral bud outgrowth

Atualmente a cultura da cana de açúcar tem ganhado destaque no cenário mundial devido a seu potencial uso na produção de bioenergia o qual poderia ser beneficiado desenvolvendo-se cultivares com aumento da produtividade de biomassa por unidade de área, o que é por sua vez, determinada pela arquitetura da planta. A brotação lateral é um dos principais fatores que regulam a arquitetura dos vegetais. Recentemente, esta fase do desenvolvimento tem sido estudada intensivamente em plantas consideradas modelo, elucidando em parte as vias genéticas, ambientais e hormonais que regulam este processo. Dentro desta vias, microRNAs, uma classe de pequenos RNAs não codantes que modula pós-transcricionalmente a expressão de genes endógenos, parecem ser importantes reguladores. Em cana de açúcar, a brotação lateral é importante para a arquitetura dos ramos laterais, germinação de gemas e perfilhamento. Entretanto, devido a sua complexidade genética e ausência de mutantes defectivos na brotação lateral, estudos nesta área ao nível molecular são limitados. Neste contexto, este trabalho teve por objetivos estudar em cana de açúcar a via microRNA156/fatores de transcrição do tipo SQUAMOSA promoter-binding-protein (SPL), a qual é associada à regulação do perfilhamento, bem como caracterizar molecularmente o processo de emergência de gemas axilares. Ferramentas computacionais usando o banco publico de ESTs TIGR gene índex permitiram identificar e classificar no genoma de cana de açúcar, diferentes genes associados ao processo de brotação lateral e resposta hormonal. Entres estes, seis genes SPL regulados pelo miR156 foram identificados, sendo um deles (SsSPL1) homólogo a SPLs envolvidas diretamente na regulação do perfilhamento. A expressão da SsSPL1 foi monitorada em diferentes tecidos e órgãos, juntamente com o miR156. Os dados sugerem que a SsPL1, é regulada negativamente pelo miR156, sendo esta via também conservada em cana de açúcar. Foram geradas plantas transgênicas da cultivar RB85486 com o gene endógeno SsmiR156a/b o qual codifica um precursor conservado do miR156 e parece estar associado com a evolução da arquitetura em monocotiledôneas. O acúmulo do miR156 foi avaliado em plantas transformadas via RT-qPCR encontrando variabilidade na sua expressão. Bibliotecas de pequenos RNAs foram geradas em gemas dormentes e em desenvolvimento, permitindo identificar membros de 26 famílias de miRNAs. A expressão de quatro deles, de seus genes-alvo e de outros genes selecionados foi monitorada em gemas dormentes e com 2 e 5 dias após o plantio. Interessantemente, o miR159 foi o mais expresso em gemas axilares de cana e parece ser um fator chave na emergência da gema, já que, segundo os resultados obtidos, esse miRNA parece modular a expressão do seu gene alvo SsGAMyB, o qual é um fator de transcrição implicado na ativação de genes de resposta a giberelina. Durante esta fase inicial do desenvolvimento também foi observado alterações na expressão de genes associados com processos de transdução de sinal associados aos fitohormônios auxina e etileno. Os resultados obtidos indicam que a emergência de gemas laterais é um processo dinâmico em que fitohormônios, fatores de transcrição e microRNAs participam conjuntamente para promover o crescimento e 12 desenvolvimento da nova plântula de cana de açúcar.Sugarcane is an economically important biofuel crop that recently has become a target for improvement of sustainable biomaterial production due to its high biomass productivity and built-in containment features. Therefore, studies aiming to improve the production of biomass per area are among the most important issues in sugarcane production. Plant biomass is defined, at least in part, by its shoot architecture. Although shoot architecture (branching/tillering) is to some extend influenced by environmental factors, it is determined mainly by the plants genetic program. This includes developmental programs that are regulated by a complex network of genetic pathways that integrate endogenous and environmental cues. Several transcription factors as well as microRNAs are likely part of this network. In this study, we started to investigate the roles of the genetic pathway regulated by the microRNA156 and its targets, the transcription factors SQUAMOSA promoter-binding-protein (SPLs) in sugarcane branching/tillering. We identified six members of the SPL family that were further classified into four subfamilies. In both dicots and monocots, these SPLs are key regulators of the plant shoot architecture. We monitored the expression patterns of SsmiR156 e SsSPL1 in distinct sugarcane tissues/organs. Our observations suggest that miR156 regulates posttranscriptionally SsSPL1 mainly in leaf tissues. We generated transgenic sugarcane plants overexpressing the monocot-specific sugarcane miR156 precursor SsMIR156b/c via biolistic method. This precursor is thought to be important for the evolution of grass shoot architecture. Although we observed higher accumulation of mature SsmiR156b/c in leaf tissues of some transgenic plants as compared with tissues from non-transgenic plants, we could not detect any significant changes in their vegetative architecture. Using deep sequencing approaches, we have generated two small RNA libraries from dormant and outgrown sugarcane lateral buds. Preliminary analyses indicate that a select group of small RNAs are expressed in lateral buds, including over 200 repeat-associated small interfering RNAs (rasiRNAs) and 25 conserved microRNAs (miRNAs). Amongst the miRNAs, miR159 was the most sequenced in the two libraries. We evaluated miR159 accumulation pattern in addition to other selected miRNAs via qRT-PCR in dormant and developing buds. The majority of the evaluated miRNAs accumulate differentially during bud development, though with distinct expression patterns. Interestingly, miR159 accumulates at high levels in dormant buds, but scarcely in developing buds. Conversely, the experimentally confirmed miR159 target, a sugarcane GAMyB-like gene (SsGAMyB), is lowly expressed in dormant buds while its transcripts accumulate at higher levels in developing buds. GAMyB-like genes encode R2R3 MYB domain transcription factors that have been implicated in gibberellin (GA) and abscisic acid (ABA) signaling in germinating seeds. Our data suggest miR159 regulates GAMyB-like genes during sugarcane bud outgrowth. Similarly, SsSPL1 is regulated posttranscriptionally by the miR529, though this gene has sites for both miR529 and miR156. Auxin and ethylene-associated regulatory pathways are affected during sugarcane bud development. Taken together,our data indicate that sugarcane bud outgrowth from rhizomes is a complex developmental process involving hormones, transcription factors as well as microRNAs and other regulatory RNAs

Papel funcional de microRNAs na arquitetura vegetativa e radicular de plantas

Os MicroRNAs (miRNAs) são pequenos RNAs endógenos não codantes de 20-22 nucleotídeos (nt) que regulam a expressão gênica de genes-alvos. Eles estão envolvidos em diversos aspectos de desenvolvimento da planta, tanto na parte aérea, quanto no sistema radicular. Os miRNAs e seus genesalvos tem uma complementariedade quase perfeita em plantas, sendo que esta complementaridade está relacionada à origem dos genes de miRNAs (genes MIRs). A hipótese mais aceita para a origem dos genes de miRNAs é a “hipótese de duplicação invertida”, a qual propõe que os genes de miRNAs surgiram a partir de duplicações invertidas do seus genes-alvos nos genomas vegetais. Muitos genes-alvos de miRNA em plantas codificam para fatores de transcrição, tais como os genes SQUAMOSA Promoter-Binding Protein-Like (SPLs). Alguns membros da família SPL são regulados por ambos os miRNAs, miR156 e miR529. O sítio de reconhecimento destes microRNAs em transcritos de genes SPLs é conservado e difere somente por sete nucleotídeos. Enquanto o miR156 é altamente conservado entre Angiospermas, incluindo arabidopsis, o miR529 aparentemente está presente somente em eudicotiledôneas basais, monocotiledôneas e no musgo Physcomitrella patens. Neste trabalho, foram realizadas duas abordagens para o estudo da via miRNAs/SPL. Na primeira abordagem, foi analisada a evolução e a possível função da via miR529/SPL em monocotiledôneas e eudicotiledôneas. Foi testado o sítio de reconhecimento do miR529b em dois genes SPLs (SPL9 e SPL15) de arabidopsis encontrados bioinformaticamente via geração de plantas transgênicas de arabidopsis superexpressando o precursor de arroz OsMIR529b. As linhagens transgênicas de arabidopsis (OsMIR529b-OE) apresentaram fenótipos vegetativos e reprodutivos similares aos de plantas duplo mutantes de perda de função para os genes SPL9 e SPL15 de arabidopsis (denominado spl9/spl15). Estes...MicroRNAs (miRNAs) are endogenous small non-coding RNAs of 20-22 nucleotides (nt) in length that regulate the gene expression transcriptionally and posttranscriptionally. They are involved in many aspects of plant development, both in the shoot and in the root systems. MiRNAs and their target genes have an almost perfect complementarily in plants, and this is related to the complementarity of genes origin of miRNAs genes (MIR genes). The most accepted hypothesis for the origin of miRNA genes is the inverted duplication hypothesis, which proposes that genes of miRNAs arose from inverted duplications of their target genes in plant genomes. Many miRNA target genes in plants encode transcription factors such as SQUAMOSA Promoter-Binding Protein-Like (SPLs). Some SPL family members SPL are regulated by both miRNAs, miR156 and miR529. The recognition site for these microRNAs in SPL transcripts is conserved and only differs by seven nucleotides. While miR156 is highly conserved among Angiosperms, including Arabidopsis thaliana, miR529 is apparently present only in basal eudicots, monocots and in the moss Physcomitrella patens. In this work, were performed two approaches to study miRNA-direct SPL gene regulation. In the first approach, we analyzed the evolution and possible function of the miR529/SPL pathway in monocots and eudicotyledonous. We searched for miR529b recognition sites in SPL genes from core eudicots, such as Arabidopsis thaliana. Interestingly, we found two miR529b-targeted SPLs (SPL9 and SPL15) in Arabidopsis and showed that the recognition sites are functional via the generation of transgenic Arabidopsis plants overexpressing OsMIR529b. OsMIR529b-OE overpressors are phenotipically and molecularly similar to the double mutant spl9/spl15. Our data suggest that miR529b is processed and functional in core eudicots and that this microRNA was recently lost in the evolutionary history of the ..