Cristina Zamora-Ballesteros

Background: Long non-coding RNAs (lncRNAs) are emerging regulators of plant immunity, but their roles in conifer-pathogen interactions remain largely unexplored. We applied a dual RNA-seq approach to resistant Pinus pinea and susceptible P. radiata challenged with Fusarium circinatum at 4 dpi, and concurrently profiled fungal lncRNAs. Results: Using a conservative multi-tool pipeline, we identified 8,783 lncRNAs in P. radiata , 5,255 in P. pinea , and 1,020 in F. circinatum . Pine lncRNAs displayed canonical features (shorter length, fewer/shorter exons, intergenic dominance) and limited primary-sequence conservation. Differential expression analysis revealed 37 ( P. radiata ) and 34 ( P. pinea ) infection-responsive lncRNAs. Predicted cis targets in P. radiata were enriched for energy/redox and gibberellin-related functions, whereas P. pinea targets pointed to TCA/redox and translation control. Weighted gene co-expression analysis placed P. radiata lncRNAs in defence-like modules without significant infection association, while P. pinea lncRNAs clustered into two modules with opposite associations to infection: one that increased and was enriched for immunity, flavonoid biosynthesis, and cell-wall processes, and another that decreased and was linked to photosynthesis and chloroplast functions. On the pathogen side, infection of the resistant host triggered distinct F. circinatum lncRNAs linked to transcriptional control and putative cis activation of cell-wall–degrading and toxin-biosynthetic genes, alongside down-regulation near ergosterol-biosynthetic loci. By contrast, during infection of the susceptible host, species-associated modules were dominated by translation-related functions. Conclusions: Together, these results define a non-coding regulatory layer that differentiates resistance and susceptibility strategies in the Pinus – F. circinatum pathosystem, provide the first lncRNA catalogues for P. pinea and F. circinatum , and deliver testable candidates for functional validation and biomarker development in forest disease management.

Mycoviruses are viruses that infect fungi, including plant pathogens. The infection of these mycoviruses is sometimes associated with impaired phenotypes of their fungal hosts, a phenomenon known as hypovirulence. Thus, using mycoviruses as biological control agents has emerged as a promising tool to combat forest diseases. The invasive ascomycete fungus Fusarium circinatum, which causes pine pitch canker (PPC) disease in Pinus tree species and other coniferous trees, is infected by the mycovirus Fusarium circinatum mitovirus 1 (FcMV1), FcMV2-1, and FcMV2-2. However, its impact on pathogen fitness remains unclear. The most accurate method used to identify the effect of a mycovirus on its host is the generation of isogenic lines with and without the mycovirus. The present study aimed to cure F. circinatum isolates infected by FcMV1 using different approaches. For this purpose, three replicates of each isolate were exposed to thermal treatment (38 °C) and antibiotic treatment (ribavirin, cycloheximide, kanamycin, and rifampicin mixed with cAMP)(cyclic adenosine monophosphate) for five successive passages. The viral titer of FcMV1 was then assessed using qPCR (quantitative polymerase chain reaction) after the first week and after the fifth week of the treatment. The results revealed differences in treatment efficacy among F. circinatum isolates, with some showing very low virus titers at the end of the experiment. Both thermal and antibiotic treatment effectively reduced the viral load in all isolates. In addition, the antibiotic cycloheximide and rifampicin +cAMP reduced the viral titer more than ribavirin and kanamycin. The isolate Fc179 was found to be more prone to antibiotic treatment than the other two isolates (001 and Va221). This study demonstrated the possibility of using some isolates of F. circinatum for fine-tuning cures for mitovirus, in order to create virus-free strains for biological control in the future.

El chancro resinoso del pino es una grave enfermedad que afecta a Pinus spp. y Pseudotsuga menziesii en todo el mundo. Varios factores han contribuido a la propagación de la enfermedad a todos estos continentes, siendo el más importante el comercio internacional de material vegetal de reproducción. A pesar de su importancia, no se dispone de medidas eficaces para erradicar o controlar la enfermedad ni en los viveros ni en el monte. En esta tesis se llevó a cabo una recopilación de información actual sobre las vías de propagación del patógeno, proponiendo mecanismos preventivos para evitar su introducción en zonas libres de la enfermedad. Proponemos más investigación sobre el posible papel de los micovirus como agentes de control biológico, explorando nuevos métodos de transfección con micovirus de F. circinatum. Utilizamos las tecnologías transcriptómicas que, además de permitirnos estudiar el efecto de un micovirus en su hongo hospedante a nivel molecular, nos proporcionaron información útil sobre los genes, las transcripciones, y sus funciones asociadas a las interacciones planta-patógeno. Mediante el uso de RNA-Seq y especies susceptibles y resistentes a F. circinatum, identificamos los genes y vías metabólicas asociados a caracteres de resistencia de la enfermedad y presentamos un mapa completo de los ARNlnc durante la infección del patógeno. Nuestros hallazgos sobre los mecanismos reguladores implicados en la defensa de los hospedantes permitirán la optimización de los programas de mejora genética para la comercialización de pinos resistentes al PPC.

Background: One of the most promising strategies of Pine Pitch Canker (PPC) management is the use of reproductive plant material resistant to the disease. Understanding the complexity of plant transcriptome that underlies the defence to the causal agentFusarium circinatum, would greatly facilitate the development of an accurate breeding program. Long non-coding RNAs (lncRNAs) are emerging as important transcriptional regulators under biotic stresses in plants. However, to date, characterization of lncRNAs in conifer trees has not been reported. In this study, transcriptomic identification of lncRNAs was carried out using strand-specific paired-end RNA sequencing, fromPinus radiatasamples inoculated withF. circinatumat an early stage of infection.Results: Overall, 13,312 lncRNAs were predicted through a bioinformatics approach, including long intergenic non-coding RNAs (92.3%), antisense lncRNAs (3.3%) and intronic lncRNAs (2.9%). Compared with protein-coding RNAs, pine lncRNAs are shorter, have lower expression, lower GC content and harbour fewer and shorter exons. A total of 164 differentially expressed (DE) lncRNAs were identified in response toF. circinatuminfection in the inoculated versus mock-inoculatedP. radiataseedlings. The predictedcis-regulated target genes of these pathogen-responsive lncRNAs were related to defence mechanisms such as kinase activity, phytohormone regulation, and cell wall reinforcement. Co-expression network analysis of DE lncRNAs, DE protein-coding RNAs and lncRNA target genes also indicated a potential network regulating pectinesterase activity and cell wall remodelling.Conclusions: This study presents the first comprehensive genome-wide analysis ofP. radiatalncRNAs and provides the basis for future functional characterizations of lncRNAs in relation to pine defence responses againstF. circinatum.

This study investigated the complex phenotypic and genetic response of Monterey pine (Pinus radiata) seedlings to co-infections by F. circinatum, the causal agent of pine pitch canker disease, and the oomycetes Phytophthora xcambivora and P. parvispora. Monterey pine seedlings were wound-inoculated with each single pathogen and with the combinations F. circinatum/P. xcambivora and F. circinatum/P. parvispora. Initially, seedlings inoculated only with F. circinatum showed less severe symptoms than seedlings co-inoculated or inoculated only with P. xcambivora or P. parvispora. However, 30 days post-inoculation (dpi), all inoculated seedlings, including those inoculated only with F. circinatum, showed severe symptoms with no significant differences among treatments. The transcriptomic profiles of three genes encoding pathogenesis-related proteins, i.e., chitinase (PR3), thaumatin-like protein (PR5), phenylalanine ammonia-lyase (PAL), and the pyruvate decarboxylase (PDC)-encoding gene were analyzed at various time intervals after inoculation. In seedlings inoculated with single pathogens, F. circinatum stimulated the up-regulation of all genes, while between the two oomycetes, only P. xcambivora induced significant up-regulations. In seedlings co-inoculated with F. circinatum and P.xcambivora or P. parvispora none of the genes showed a significant over-expression 4 dpi. In contrast, at 11 dpi, significant up-regulation was observed for PR5 in the combination F. circinatum/P.xcambivora and PDC in the combination F. circinatum/P. parvispora, thus suggesting a possible synergism of multiple infections in triggering this plant defense mechanism.

One of the most promising strategies of Pine Pitch Canker (PPC) management is the use of reproductive plant material resistant to the disease. Understanding the complexity of plant transcriptome that underlies the defence to the causal agent Fusarium circinatum , would greatly facilitate the development of an accurate breeding program. Long non-coding RNAs (lncRNAs) are emerging as important transcriptional regulators under biotic stresses in plants. However, to date, characterization of lncRNAs in conifer trees has not been reported. In this study, transcriptomic identification of lncRNAs was carried out using strand-specific paired-end RNA sequencing, from Pinus radiata samples inoculated with F. circinatum at an early stage of infection. Overall, 13,312 lncRNAs were predicted through a bioinformatics approach, including long intergenic non-coding RNAs (92.3%), antisense lncRNAs (3.3%) and intronic lncRNAs (2.9%). Compared with protein-coding RNAs, pine lncRNAs are shorter, have lower expression, lower GC content and harbour fewer and shorter exons. A total of 164 differentially expressed (DE) lncRNAs were identified in response to F. circinatum infection in the inoculated versus mock-inoculated P. radiata seedlings. The predicted cis -regulated target genes of these pathogen-responsive lncRNAs were related to defence mechanisms such as kinase activity, phytohormone regulation, and cell wall reinforcement. Co-expression network analysis of DE lncRNAs, DE protein-coding RNAs and lncRNA target genes also indicated a potential network regulating pectinesterase activity and cell wall remodelling. This study presents the first analysis of conifer lncRNAs involved in the regulation of defence network and provides the basis for future functional characterizations of lncRNAs in relation to pine defence responses against F. circinatum .

Fusarium circinatum causes one of the most important diseases of conifers worldwide, the pine pitch canker (PPC). However, no effective field intervention measures aiming to control or eradicate PPC are available. Due to the variation in host genetic resistance, the development of resistant varieties is postulated as a viable and promising strategy. By using an integrated approach, this study aimed to identify differences in the molecular responses and physiological traits of the highly susceptible Pinus radiata and the highly resistant Pinus pinea to F. circinatum at an early stage of infection. Dual RNA-Seq analysis also allowed to evaluate pathogen behavior when infecting each pine species. No significant changes in the physiological analysis were found upon pathogen infection, although transcriptional reprogramming was observed mainly in the resistant species. The transcriptome profiling of P. pinea revealed an early perception of the pathogen infection together with a strong and coordinated defense activation through the reinforcement and lignification of the cell wall, the antioxidant activity, the induction of PR genes, and the biosynthesis of defense hormones. On the contrary, P. radiata had a weaker response, possibly due to impaired perception of the fungal infection that led to a reduced downstream defense signaling. Fusarium circinatum showed a different transcriptomic profile depending on the pine species being infected. While in P. pinea, the pathogen focused on the degradation of plant cell walls, active uptake of the plant nutrients was showed in P. radiata. These findings present useful knowledge for the development of breeding programs to manage PPC.

Mycoviruses are known to be difficult to cure in fungi but their spontaneous loss occurs commonly. The unexpected disappearance of mycoviruses can be explained by diverse reasons, from methodological procedures to biological events such as posttranscriptional silencing machinery. The long-term effects of a virus infection on the host organism have been well studied in the case of human viruses; however, the possible residual effect on a fungus after the degradation of a mycovirus is unknown. For that, this study analyses a possible residual effect on the transcriptome of the pathogenic fungus Fusarium circinatum after the loss of the mitovirus FcMV1. The mycovirus that previously infected the fungal isolate was not recovered after a 4-year storage period. Only 14 genes were determined as differentially expressed and were related to cell cycle regulation and amino acid metabolism. The results showed a slight acceleration in the metabolism of the host that had lost the mycovirus by the upregulation of the genes involved in essential functions for fungal development. The analysis also revealed a weak expression in the annotated genes of the RNA silencing machinery. To our knowledge, this is the first time that a potential residual effect on the host transcriptome caused by the past infection of a mycovirus is reported.

Fusarium circinatum, the causal agent of pine pitch canker (PPC), is currently one of the most important threats of Pinus spp. globally. This pathogen is known in many pine-growing regions, including natural and planted forests, and can affect all life stages of trees, from emerging seedlings to mature trees. Despite the importance of PPC, the global distribution of F. circinatum is poorly documented, and this problem is also true of the hosts within countries that are affected. The aim of this study was to review the global distribution of F. circinatum, with a particular focus on Europe. We considered (1) the current and historical pathogen records, both positive and negative, based on confirmed reports from Europe and globally; (2) the genetic diversity and population structure of the pathogen; (3) the current distribution of PPC in Europe, comparing published models of predicted disease distribution; and (4) host susceptibility by reviewing literature and generating a comprehensive list of known hosts for the fungus. These data were collated from 41 countries and used to compile a specially constructed geo-database. A review of 6297 observation records showed that F. circinatum and the symptoms it causes on conifers occurred in 14 countries, including four in Europe, and is absent in 28 countries. Field observations and experimental data from 138 host species revealed 106 susceptible host species including 85 Pinus species, 6 non-pine tree species and 15 grass and herb species. Our data confirm that susceptibility to F. circinatum varies between different host species, tree ages and environmental characteristics. Knowledge on the geographic distribution, host range and the relative susceptibility of different hosts is essential for disease management, mitigation and containment strategies. The findings reported in this review will support countries that are currently free of F. circinatum in implementing effective procedures and restrictions and prevent further spread of the pathogen.