Juan Diego Astacio

Brown rot is a disease that affects stone and pome fruit crops worldwide. It is caused by fungal members of the genus Monilinia, mainly M. fructicola, M. laxa, and M. fructigena. This study presents evidence that, despite having a very similar battery of cell wall-degrading enzymes, the three species behave differently during the early stages of infection, suggesting differences at the regulatory level, which could also explain the differences in host preference among the three species. We have shown that M. fructicola infection is accelerated by red light, and the first symptoms appear much earlier than in darkness or in the other two species. The overexpression of genes encoding for CAZymes, such as pme3, pme2, pg1, cel1, pnl1, and pnl2, as well as the necrosis factor nep2, can be associated with the etiology of Monilinia spp. In addition, we found that nep2 in M. fructigena lacks binding sites in its promoter sequence for the white-collar complex, which is the major transcription factor responsible for regulating photoreception processes in fungi. Finally, we found that AlphaFold models of the NEP1-like proteins present on the three Monilinia species predict proteins with a very high degree of similarity.

Brown rot is the main fungal disease affecting stone fruit crops worldwide. It is caused by fungi of the genus Monilinia, which are a filamentous fungi from the Sclerotiniaceae family with a necrotrophic lifestyle. They employ a number of virulence factors to penetrate the plant cell wall and membranes. The development of brown rot is controlled by environmental factors and the maturity stage of the host. In recent years, lighting conditions are gaining relevance as an environmental factor that can modify the course of development of the disease caused by Monilinia spp. The aim of this thesis is to study how different wavelengths and photoperiods affect the host-pathogen-environment interaction. For this purpose, molecular and bioinformatics techniques were used to identify the photoinducible genes, as well as those coding for virulence factors and photoreceptors in each Monilinia species. Growth assays were performed on different synthetic liquid media in addition to infection assays on fruit to quantify the effect of different wavelengths and photoperiods on fungal growth, disease development, and fungal transcriptional response. The three Monilinia species encode a very similar set of CAZymes in their respective genomes. A total of 414, 398, and 383 CAZymes were identified in the genomes of M. fructicola 38C, M. laxa 8L, and M. fructigena Mgena6, respectively. In addition, two genes coding for NEP1-like toxins were found in the genomes of the three Monilinia species. Red and blue lights caused an increase in the transcriptional activity of M. fructicola 38C virulence factors during the first hours of the infection process on nectarine, which was accompanied by a reduction of the latency period of the disease. In the case of M. laxa 8L, fewer genes also experienced photoinduction by red light. In M. fructigena Mgena6 photoinduction was residual, while some virulence factors saw their gene expression levels increased under dark conditions. No light-caused differences in the development of disease symptoms under our experimental conditions were observed in either M. laxa or M. fructigena. On the other hand, M. fructicola 38C showed differences in gene expression levels of CAZymes and Mfcnep2 when grown on different synthetic culture media and on fruit. There are differences in daily growth rate and sporulation at the species level when the fungus is grown on solid medium and illuminated by different types of light and photoperiods. Red light produced an increase in the daily growth rate in M. fructicola 38C, while all other wavelengths reduced it. M. fructigena Mgena6 showed a reduction in growth rate in response to all wavelengths under a short photoperiod, while under a long photoperiod white, green and near ultraviolet lights reduced it significantly. All three Monilinia species have in their genome information to encode the same set of photoreceptor proteins. This set is composed of three phytochromes, two cryptochromes, three LOVs, and two rhodopsins. This thesis shows the existence of mechanisms that allow differential regulation by light of disease development and virulence factors in each species. Despite having identical photoreceptor machinery, we observed differences in light-responsive behavior among the three Monilinia species, including an increase in the rate of appearance of brown rot symptoms by M. fructicola 38C and increased transcriptional activity of several virulence factors. RESUMEN La podredumbre parda es una de las principales enfermedades fúngicas que afecta al cultivo de fruta de hueso en todo el mundo. Está causada por hongos del género Monilinia de la familia Sclerotiniaceae, con un estilo de vida necrotrofo, que emplea una serie de factores de virulencia para atravesar la pared y membranas celulares vegetales y causar la muerte de éstas para alimentarse de su contenido. El desarrollo de la podredumbre parda está controlado por factores ambientales y el estado de maduración del huésped. En los últimos años las condiciones de iluminación están cobrando relevancia como factor ambiental que puede alterar el desarrollo de la enfermedad causada por Monilinia spp. Esta tesis se plantean cuatro objetivos para estudiar cómo afectan distintas longitudes de onda y fotoperiodos a la interacción huésped-patógeno-ambiente. Para ello, se usaron técnicas moleculares y bioinformáticas para identificar los genes fotoinducibles, así como los codificantes para factores de virulencia y fotorreceptores en cada especie de Monilinia. Se realizaron ensayos de crecimiento sobre distintos medios líquidos sintéticos además de ensayos de infección sobre nectarina para poder cuantificar el efecto de distintas longitudes de onda y fotoperiodos en el crecimiento del hongo, desarrollo de la enfermedad, y en la respuesta transcripcional del hongo. Las tres especies de Monilinia codifican en sus respectivos genomas un set de CAZymes muy similar. Se identificaron un total de 414, 398 y 383 CAZymes en los genomas de M. fructicola 38C, M. laxa 8L y M. fructigena Mgena6, respectivamente. Además, en el genoma de las tres especies de Monilinia se localizan dos genes codificantes para toxinas NEP1-like. Las luces roja y azul provocaron un aumento en la actividad transcripcional de los genes Mfcpme3, Mfcpme2, Mfcpg1, Mfccel1, Mfcpnl1, Mfcpnl2 y Mfcnep2 en M. fructicola 38C durante las primeras horas del proceso de infección sobre nectarina, lo cual vino acompañado por una reducción del periodo de latencia de la enfermedad. En el caso de M. laxa 8L, una menor cantidad de genes también experimentaron fotoinducción por luz roja. En M. fructigena Mgena6 la fotoinducción fue residual, mientras que algunos factores de virulencia vieron sus niveles de expresión génica aumentados en condiciones de oscuridad. No se observaron diferencias causadas por...

Light represents a powerful signal for the regulation of virulence in many microbial pathogens. Monilinia fructicola is the most virulent species causing brown rot in stone fruit crops. To understand the influence of light on M. fructicola, we measured the effect of white light and photoperiods on the colonial growth and sporulation of the model M. fructicola strain 38C on solid cultures. Searches in the M. fructicola 38C genome predicted a complete set of genes coding for photoreceptors possibly involved in the perception of all ranges of wavelengths. Since white light had an obvious negative effect on vegetative growth and the asexual development of M. fructicola 38C on potato dextrose agar, we studied how light influences photoresponse genes in M. fructicola during early peach infection and in liquid culture. The transcriptomes were analyzed in “Red Jim” nectarines infected by M. fructicola 38C and subjected to light pulses for 5 min and 14 h after 24 h of incubation in darkness. Specific light-induced genes were identified. Among these, we confirmed in samples from infected fruit or synthetic media that blue light photoreceptor vvd1 was among the highest expressed genes. An unknown gene, far1, coding for a small protein conserved in many families of Ascomycota phylum, was also highly induced by light. In contrast, a range of well-known photoreceptors displayed a low transcriptional response to light in M. fructicola from nectarines but not on the pathogen mycelium growing in liquid culture media for 6 days.