Eventos tempranos en el proceso de infección de Macrophomina phaseolina (Tassi) Goid. en raíces de plántulas de Pinus radiata D. Don y sus efectos en la eficiencia fotosintética como indicador de estrés biótico

Early events occurring after inoculation of Pinus radiata seedlings with Macrophomina phaseolina, fungal pathogen causing charcoal root rot, are unknown. The objectives of this study were to determine the germination process of microsclerotia and penetration of hyphae of M. phaseolina into roots of P. radiata seedlings and to establish probably effects on photosynthetic efficiency during early hours of infection pathogen. For this, 25-day-old seedling roots were inoculated with M. phaseolinamicroesclerotia under in vitro conditions. First hours of disease cycle after root inoculation were studied by scanning electron microscopy and efficiency of photosystem II by measuring the fluorescence of chlorophyll a, after inoculating of P. radiata seedling roots with M. phaseolina microesclerotia. The penetration of the pathogen into the root occurred 48 h post inoculation (hpi), and surface colonization was evident by the presence of numerous hyphae after 72 hpi. The parameters of chlorophyll a fluorescence showed variations at once occurred colonization and penetration of seedling roots, with a decrease of Fv/ Fm values from 120 hpi. Infection root of P. radiata with M. phaseolina microesclerotia generated a stress in the plant which resulted in variations in fluorescence parameters of chlorophyll a.Los eventos tempranos que ocurren luego de inocular raíces de plántulas de Pinus radiata con el hongo patógeno Macrophomina phaseolina son desconocidos. Los objetivos de este estudio fueron conocer el proceso de germinación de los microesclerocios y la penetración de las hifas de M. phaseolina en raíces de plántulas de P. radiata, y establecer posibles efectos en la eficiencia fotosintética durante las primeras horas de la infección del patógeno. Para ello se inocularon raíces de plántulas de 25 días de edad con microesclerocios de M. phaseolina bajo condiciones in vitro. Las primeras horas del ciclo de enfermedad, seguido a la inoculación de las raíces, fueron estudiadas mediante microscopía electrónica de barrido y la eficiencia del fotosistema II mediante la fluorescencia de la clorofila a. La penetración del patógeno al interior de la raíz ocurrió 48 h post-inoculación (hpi), y la colonización de la superficie de la raíz se hizo evidente por la presencia de numerosas hifas una vez transcurridas 72 hpi. Los parámetros de fluorescencia de la clorofila a evidenciaron variaciones una vez ocurrida la colonización y la penetración de las raíces de las plántulas, con una disminución de los valores Fv/Fm a partir de 120 hpi. La infección de la raíz de P. radiata causada por M. phaseolina provocó un estrés en la planta, evidenciado por una variación de los parámetros de fluorescencia de la clorofila a

Respuesta transcripcional temprana de los genes SCL y COMT y disminución de la eficiencia del fotosistema II en plántulas de Pinus radiata frente a infecciones con Macrophomina phaseolina

En la interacción planta-patógeno, tras el reconocimiento del patógeno por parte del hospedero, se activa la señalización intracelular y la expresión de factores de transcripción que regulan las respuestas celulares de defensa. Entre estos factores de transcripción se encuentran genes maestros que activan otros genes de expresión tardía. Conocer estos mecanismos moleculares aportará información base a los programas de mejoramiento genético para el desarrollo de estrategias orientadas a obtener variedades o clones más resistentes a las enfermedades. El estudio del transcriptoma nos permite conocer que genes se expresan después de un ataque por patógenos y el patrón de expresión en diferentes tiempos. En la actualidad, existe la posibilidad de que algunas enfermedades emergentes aumenten su incidencia y prevalencia; tal es el caso del patógeno de suelo Macrophomina phaseolina causante de la podredumbre carbonosa. En esta tesis se pretenden conocer eventos tempranos de la interacción de raíces de Pinus radiata con el patógeno Macrophomina phaseolina, que permitirán en futuros estudios acotar y refinar la búsqueda de genes en etapas concretas de la interacción. Por otra parte, se estudió como la inoculación del patógeno compromete la fisiología de las plántulas mediante la medida de la eficiencia del fotosistema II en las acículas de Pinus radiata. Finalmente, se evaluó la expresión relativa de los genes SCL y COMT durante las etapas tempranas de la interacción. Este trabajo es el primer reporte mediante microscopía electrónica de barrido de los eventos tempranos ocurridos tras la inoculación de plántulas de una especie de interés forestal como es Pinus radiata, con el patógeno Macrophomina phaseolina. La penetración del patógeno al interior de la raíz se produce alrededor de las 48 horas post-inoculación (hpi), siendo evidente la colonización superficial y numerosos puntos de penetración una vez transcurridas las 72 hpi. La eficiencia del fotosistema II muestra variaciones una vez ocurridos estos sucesos, concretamente una disminución de los valores Fv/Fm a partir de las 120 hpi. Los resultados demuestran la virulencia del patógeno a las pocas horas una vez ocurrido el contacto con la raíz, y como este hecho provoca un estrés fisiológico en la planta. A pesar de lo anteriormente descrito, a nivel transcripcional no se observaron diferencias significativas en la acumulación de los transcritos correspondientes a los genes SCL y COMT en plántulas inoculadas

Genome-Wide Analysis of Somatic Embryogenesis-Related Transcription Factors in Cultivated Strawberry (Fragaria × ananassa) and Evolutionary Relationships among Rosaceae Species

Somatic embryogenesis is a plant regeneration method commonly used in tissue culture. Its molecular mechanisms are well-known in model plants such as Arabidopsis thaliana L. LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON2 (LEC2), FUSCA3 (FUS3), ABSCISIC ACID INSENSITIVE3 (ABI3), and BABYBOOM (BBM) genes are considered master regulators in the induction, growth, and maturation of somatic embryos. However, the study of these transcription factors in fruit crops with high agronomic and economic value such as cultivated strawberry (Fragaria × ananassa Duch.) and other Rosaceae species is scarce. The purpose of this study was the in silico characterization of LEC1, ABI3, FUS3, LEC2, and BBM(LAFL-B) genes from F. × ananassa genome and the study of the evolutionary relationships within the Rosaceae family. Synteny analyses and molecular evolutionary rates were performed to analyze the evolution of each transcription factor within the Rosaceae family. Synteny was conserved between F. × ananassa and other Rosaceae genomes, and paralogous genes were selected through negative selection. Additionally, the exon–intron organization and multiple alignments showed that gene structure and DNA-binding domains were conserved in F. × ananassa transcription factors. Finally, phylogenetic trees showed close evolutionary relationships between F. × ananassa and its orthologous proteins in the Rosoideae subfamily. Overall, this research revealed novel insights in the LAFL-B network in F. × ananassa and other species of the Rosaceae family. These results provide useful in silico information and new resources for the establishment of more efficient propagation systems or the study of ploidy effects on somatic embryogenesis

Evolutionary Analysis of JAZ Proteins in Plants: An Approach in Search of the Ancestral Sequence

Jasmonates are phytohormones that regulate development, metabolism and immunity. Signal transduction is critical to activate jasmonate responses, but the evolution of some key regulators such as jasmonate-ZIM domain (JAZ) repressors is not clear. Here, we identified 1065 JAZ sequence proteins in 66 lower and higher plants and analyzed their evolution by bioinformatics methods. We found that the TIFY and Jas domains are highly conserved along the evolutionary scale. Furthermore, the canonical degron sequence LPIAR(R/K) of the Jas domain is conserved in lower and higher plants. It is noteworthy that degron sequences showed a large number of alternatives from gymnosperms to dicots. In addition, ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motifs are displayed in all plant lineages from liverworts to angiosperms. However, the cryptic MYC2-interacting domain (CMID) domain appeared in angiosperms for the first time. The phylogenetic analysis performed using the Maximum Likelihood method indicated that JAZ ortholog proteins are grouped according to their similarity and plant lineage. Moreover, ancestral JAZ sequences were constructed by PhyloBot software and showed specific changes in the TIFY and Jas domains during evolution from liverworts to dicots. Finally, we propose a model for the evolution of the ancestral sequences of the main eight JAZ protein subgroups. These findings contribute to the understanding of the JAZ family origin and expansion in land plants

Interactions of JAZ repressors with anthocyanin biosynthesis-related transcription factors of fragaria × ananassa

© 2020 by the authors.Strawberry fruits are rich in flavonoids like proanthocyanidins and anthocyanins. Their biosynthesis and accumulation are controlled by the MYB-bHLH-WD40 (MBW) transcriptional complex, which is mainly formed by basic helix-loop-helix (bHLH) and MYB transcription factors (TFs). In Arabidopsis thaliana both bHLH and MYB TFs are repressed by JASMONATE ZIM-DOMAIN (JAZ) proteins, the key repressors of the jasmonate-signaling pathway. The aim of this research was the characterization of the FaJAZ1/8.1/9/10 proteins and molecular targets of signaling components and anthocyanin biosynthesis-related TFs of Fragaria × ananassa by protein–protein interactions. For this, domain compositions were studied by multiple alignments and phylogenetic analyses, while interactions were analyzed by yeast two-hybrid (Y2H) assays. We detected high conservation of FaJAZ proteins and jasmonate-signaling components, as well as FabHLHs and FaMYB10 TFs. Moreover, we report the F. × ananassa YABBY1 (FaYAB1) TF, which is related to anthocyanin biosynthesis in Arabidopsis, showed high conservation of functional domains. We demonstrated that FaJAZ repressors interacted with F. × ananassa NOVEL INTERACTOR OF JAZ (FaNINJA), FaMYC2, and JASMONATE ASSOCIATED MYC2-LIKE (FaJAM) proteins. Besides, transcription factors of MBW-complex like FabHLH3, FabHLH33, and FaMYB10, together with FaYAB1, were molecular targets of FaJAZ repressors, exhibiting specificity or redundancy of interaction depending on particular FaJAZ protein. Overall, these results suggest that interactions of jasmonate-signaling components are fully conserved, and anthocyanin biosynthesis might be regulated by JAZ repressors in F. × ananassa.This research was funded by the National Research and Development Agency (ANID, Chile) grant FONDECYT/Regular 1181310 to C.R.F. Work in R.S.’s laboratory was funded by the Spanish Ministry of Science, Innovation and Universities grant BIO2019-107012RB (MICINN/FEDER). A.G.-B. and M.T. acknowledge ANID, grants FONDECYT/Postdoctorado 3190894, and FONDECYT/Regular 1180926, respectively

Correction: Jasmonate signalling pathway in strawberry: Genome-wide identification, molecular characterization and expression of JAZs and MYCs during fruit development and ripening.

[This corrects the article DOI: 10.1371/journal.pone.0197118.]

A new functional JAZ degron sequence in strawberry JAZ1 revealed by structural and interaction studies on the COI1–JA-Ile/COR–JAZs complexes

© The Author(s) 2020.The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates fundamental plant processes as developmental and defense responses. JA-Ile mediates the interaction between the F-box protein COI1 (part of the SCF E3 ubiquitin ligase) and a JAZ repressor leading to early jasmonate responses. The Arabidopsis JAZ1 protein contains the canonical LPIARR degron sequence, which is responsible for the stabilization of the AtCOI1-JA-Ile-AtJAZ1 complex. In strawberry (Fragaria × ananassa) JAZ family was described at the transcriptional level during fruit development but the information about the interaction mode of this complex is still scarce at the molecular level. To gain insight into the strawberry JA-Ile receptor complex, we evaluated the interaction at the structural level, and protein models were built and analyzed for FaCOI1 and FaJAZ1, FaJAZ8.1, and FaJAZ10. The interaction between FaCOI1 and FaJAZ1, FaJAZ8.1 and FaJAZ10 were explored using several ligands, through molecular docking and molecular dynamics (MD) simulations, finding the strongest interaction with (+)-7-iso-JA-Ile than other ligands. Additionally, we tested interactions between FaCOI1 and FaJAZs by yeast two-hybrid assays in the presence of coronatine (COR, a JA-Ile mimic). We detected strong COR-dependent interactions between FaCOI1 and FaJAZ1. Interestingly, FaJAZ1 contains a new non-canonical (IPMQRK) functional degron sequence, in which Arg and Lys are the key residues for maintaining the interaction of the FaCOI1–COR–FaJAZ1 complex as we observed in mutated versions of the FaJAZ1 degron. Phylogenetic analysis showed that the IPMQRK degron is only present in orthologs belonging to the Rosoideae but not in other Rosaceae subfamilies. Together, this study uncovers a new degron sequence in plants, which could be required to make an alternative and functional JA-Ile perception complex in strawberry.This research was funded by the National Research and Development Agency (ANID, Chile), grant FONDECYT/Regular 1181310 to Carlos R. Figueroa. Adrián Garrido-Bigotes acknowledges ANID (grant FONDECYT/Postdoctorado 3190894). F.V-R acknowledges Universidad de Talca for a doctoral scholarship. Work in R. Solano’s laboratory was funded by the Spanish Ministry of Economy and Competitivity grant BIO2016-77216-R (MINECO/FEDER)

Jasmonate signalling pathway in strawberry: Genome-wide identification, molecular characterization and expression of <i>JAZ</i>s and <i>MYC</i>s during fruit development and ripening

<div><p>Jasmonates (JAs) are signalling molecules involved in stress responses, development and secondary metabolism biosynthesis, although their roles in fleshy-fruit development and ripening processes are not well known. In strawberry fruit, it has been proposed that JAs could regulate the early development through the activation of the JAs biosynthesis. Moreover, it has been reported that JA treatment increases anthocyanin content in strawberry fruit involving the bioactive jasmonate biosynthesis. Nevertheless, JA signalling pathway, of which main components are the COI1-JAZ co-receptor and the MYC transcription factors (TFs), has not been characterized in strawberry until now. Here we identified and characterized the woodland strawberry (<i>Fragaria vesca</i>) JAZ and MYC genes as well as studied their expression during development and ripening stages in commercial strawberry (<i>Fragaria</i> × <i>ananassa</i>) fruit. We described twelve putative JAZ proteins and two MYC TFs, which showed high conservation with respect to their orthologs in <i>Arabidopsis thaliana</i> and in other fleshy-fruit species such as <i>Malus</i> × <i>domestica</i>, <i>Vitis vinifera</i> and <i>Solanum lycopersicum</i> as revealed by gene synteny and phylogenetic analyses. Noteworthy, their expression levels exhibited a significant decrease from fruit development to ripening stages in <i>F</i>. × <i>ananassa</i>, along with others of the JA signalling-related genes such as <i>FaNINJA</i> and <i>FaJAM</i>s, encoding for negative regulators of JA responses. Moreover, we found that main JA signalling-related genes such as <i>FaMYC2</i>, and <i>FaJAZ1</i> are promptly induced by JA treatment at early times in <i>F</i>. × <i>ananassa</i> fruit. These results suggest the conservation of the canonical JA signalling pathway in strawberry and a possible role of this pathway in early strawberry fruit development, which also correlates negatively with the beginning of the ripening process.</p></div

Exon-intron structures of the <i>Fragaria vesca</i> and Arabidopsis <i>JAZ</i> and <i>MYC</i> genes.

<p>Exon-intron organization of <i>JAZ</i> genes (A) and <i>MYC</i> genes (B) in <i>F</i>. <i>vesca</i> (Fv) and <i>Arabidopsis thaliana</i> (At) grouped according their gene orthology. Yellow and red bars indicate untranslated (UTR) regions and exons, respectively. Black interrupted lines indicate introns. JAZ, jasmonate ZIM-domain.</p