Caracterización de potenciales efectores fitopatogénicos del fitoplasma del grupo 16SrXIII-F “Strawberry phyllody phytoplasma Chilean strain”

Abstract

Los fitoplasmas son patógenos intracelulares no cultivables capaces de colonizar células eucariontes vegetales y animales. Su transmisión es persistente y propagativa, causando síntomas en plantas como enanismo, filodia y escoba de brujas. Esto disminuye la productividad de cultivos de interés comercial a nivel global y local. En especifico, el fitoplasma “Strawberry Phyllody Chile” (StrPh-CL) es una bacteria que se asocia a la aparición de síntomas de filodia en cultivos de frutilla, además de afectar cultivos tanto comerciales como nativos. Pertenece al grupo ribosomal 16SrXIII, nativo de América, y corresponde al primer fitoplasma de este grupo cuyo genoma está secuenciado. En el genoma del fitoplasma StrPh-CL se identificaron 25 genes que codifican candidatos de efectores patogénicos, entre los cuales se encuentran homólogos de TENGU y SAPs, efectores “clásicos” de virulencia en plantas. No se han realizado estudios que describan los mecanismos moleculares de virulencia de fitoplasmas del grupo 16SrXIII, por lo que se seleccionaron los genes homólogos a Tengu y SAPs de StrPh-CL para evaluar la inducción de síntomas en hospederos vegetales. Se realizó la transmisión de StrPh-CL a la planta modelo Arabidopsis thaliana (A. thaliana) mediante insectos vectores (Cixiosoma sp.) obtenidos en cultivos de frutilla de la VI Región del Libertador General Bernardo O’Higgins. Los síntomas de infección de StrPh-CL observados en A. thaliana son disminución de la altura de la planta, aumento del área foliar, retraso del desarrollo floral y elongación de tallo. Este es el primer reporte que describe los síntomas de StrPh-CL en plantas modelo, proporcionando información valiosa para futuros estudios in vivo de fitoplasmas del grupo ribosomal 16SrXIII. Para determinar la capacidad de los genes que codifican los potenciales efectores de causar síntomas en plantas, se construyeron tres vectores virales recombinantes para la expresión heteróloga individual de los candidatos a efectores patogénicos en Nicotiana benthamiana. Se realizó transcripción in vitro con el sistema mMESSAGE mMACHINE™ T7 de los vectores virales mutantes pBSG1057-tengu strphCL, pBSG1057-sap54 - strphCL, pBSG1057-sap42 strphCL, y pBSG1057-gfp. Luego, se inocularon en plantas de N. benthamiana de 4 semanas de edad mediante transmisión mecánica. Para cada vector, se inocularon 8 plantas. Del total de 32 plantas inoculadas, sólo se logró la infección en un individuo con un virus recombinante que expresa sap42 strphCL, que presentó síntomas de enanismo, alteraciones en la morfología floral, aparición de zarcillos, hojas rugosas y ampollas foliares. A partir de los síntomas observados, se puede concluir que SAP42 puede causar alteraciones en el crecimiento y desarrollo floral en planta, contribuyendo a la virulencia del fitoplasma actuando como efector fitopatogénico. Esto propone la existencia de una nueva familia de efectores patogénicos que podrían explicar la virulencia de fitoplasmas del grupo 16SrXIII, que sólo se encuentran en América.

Phytoplasmas are unculturable intracellular pathogens capable of colonizing eukaryotic plant and animal cells. Their transmission is persistent and propagative between plants and insects; causing symptoms in plant hosts such as dwarfism, phyllody, witches’ broom, and general decline of individuals, which decreases the productivity of commercially significant crops at both global and local levels. Specifically, the phytoplasma “Strawberry Phyllody Chile” (StrPh-CL) is a bacterium associated with the appearance of phyllody symptoms in strawberry crops, in addition to affecting other commercial and native plant hosts. It belongs to the 16SrXIII ribosomal group “Mexican Periwinkle Virescence Group” and is the first native phytoplasma of this group native to the Americas with a fully sequenced genome. Phytoplasmas colonize their plant hosts by secreting pathogenic effectors through the Sec pathway. These pathogenic effectors are directly related to the pathogen’s virulence, with four effector families identified so far as symptom inducers in plants. In the genome of the StrPh-CL phytoplasma, 25 candidate genes for pathogenic effectors were identified, including homologs of TENGU and SAPs, “classic” phytoplasma pathogenic effectors. To date, no studies have described the molecular mechanisms of virulence of phytoplasmas from the 16S rRNAXIII group; therefore, these StrPh-CL genes were selected to evaluate their contribution to virulence in plant hosts, providing valuable information for future in vivo studies of phytoplasmas from the 16S rRNAXIII ribosomal group. Transmission of StrPh-CL to the model plant Arabidopsis thaliana (A. thaliana) was carried out using vector insects (Cixiosoma sp.) carrying phytoplasma obtained from strawberry crops in the VI Region of Libertador General Bernardo O’Higgins. Symptoms of StrPh-CL infection observed in A. thaliana include reduced plant height, increased leaf area, delayed floral development, and stem elongation. This is the first report describing StrPh-CL symptoms in model plants, providing valuable information for future in vivo studies of phytoplasmas from the 16SrXIII ribosomal group. To determine the individual capacity of the candidate pathogenic effectors encoded in the StrPh-CL genome to cause symptoms in plants, recombinant viral vectors from an attenuated strain of Tobacco mosaic Virus (pBSG1057) were constructed. The goal was to replicate the StrPh-CL infection phenotype by heterologous expression of the candidate pathogenic effectors in Nicotiana benthamiana (N. benthamiana). In vitro transcription was performed using the mMESSAGEmMACHINE™ T7 system for the mutant viral vectors pBSG1057-tengu strphCL, pBSG1057- sap54 strphCL, pBSG1057-sap42 strphCL, and pBSG1057-gfp. These vectors were then individually inoculated into 4-week-old N. benthamiana plants via mechanical transmission. For each vector, 8 plants were inoculated. Out of the 32 inoculated plants, only one individual was successfully infected with pBSG1057- sap42 strphCL, which showed symptoms 2 weeks post-inoculation that persisted until the plant’s death. Symptoms included dwarfism, alterations in floral morphology, tendril formation, rough leaves, and foliar blisters. Based on the observed symptoms, it can be concluded that SAP42 can cause alterations in plant growth and floral development, acting as a pathogenic effector. This is the first report associating SAP42 with plant symptomatology, proposing a whole new family of phytopathogenic effectors that could explain the virulence of 16S rRNAXIII group phytoplasmas, which are found only in the Americas.