Kinetic Study of the Avocado Sunblotch Viroid Self-Cleavage Reaction Reveals Compensatory Effects between High-Pressure and High-Temperature: Implications for Origins of Life on Earth

A high pressure apparatus allowing one to study enzyme kinetics under pressure was used to study the self-cleavage activity of the avocado sunblotch viroid. The kinetics of this reaction were determined under pressure over a range up to 300 MPa (1–3000 bar). It appears that the initial rate of this reaction decreases when pressure increases, revealing a positive ΔV≠ of activation, which correlates with the domain closure accompanying the reaction and the decrease of the surface of the viroid exposed to the solvent. Although, as expected, temperature increases the rate of the reaction whose energy of activation was determined, it appeared that it does not significantly influence the ΔV≠ of activation and that pressure does not influence the energy of activation. These results provide information about the structural aspects or this self-cleavage reaction, which is involved in the process of maturation of this viroid. The behavior of ASBVd results from the involvement of the hammerhead ribozyme present at its catalytic domain, indeed a structural motif is very widespread in the ancient and current RNA world.

A Highly Sensitive Method to Detect Avocado Sunblotch Viroid for the Maintenance of Infection-Free Avocado Germplasm Collections

The United States Department of Agriculture (USDA) Agricultural Research Service (ARS) Subtropical Horticulture Research Station (SHRS) in Miami, FL holds a large germplasm collection of avocado (Persea americana). The recent threat of infection by laurel wilt has encouraged the creation of a backup collection at a disease-free site. Creating the backup collection is complicated by infection of some trees in the germplasm collection with avocado sunblotch viroid (ASBVd). Infected trees are frequently asymptomatic, necessitating the use of a molecular diagnostic assay. Although a reverse-transcription based assay already exists and has been used to assay all germplasm at the station, some trees showed inconsistent results. We have developed a more sensitive and specific assay involving pre-amplification of the entire viroid cDNA followed by detection using real-time PCR and a TaqMan assay. A second screening of all germplasm identified additional ASBVd -infected trees and allowed us to confidently remove these trees from the station. This method enables avocado germplasm curators to proceed with the creation of a viroid-free backup collection.

The Avocado Sunblotch Viroid: An Invisible Foe of Avocado

This review collects information about the history of avocado and the economically important disease, avocado sunblotch, caused by the avocado sunblotch viroid (ASBVd). Sunblotch symptoms are variable, but the most common in fruits are irregular sunken areas of white, yellow, or reddish color. On severely affected fruits, the sunken areas may become necrotic. ASBVd (type species Avocado sunblotch viroid, family Avsunviroidae) replicates and accumulates in the chloroplast, and it is the smallest plant pathogen. This pathogen is a circular single-stranded RNA of 246–251 nucleotides. ASBVd has a restricted host range and only few plant species of the family Lauraceae have been confirmed experimentally as additional hosts. The most reliable method to detect ASBVd in the field is to identify symptomatic fruits, complemented in the laboratory with reliable and sensitive molecular techniques to identify infected but asymptomatic trees. This pathogen is widely distributed in most avocado-producing areas and causes significant reductions in yield and fruit quality. Infected asymptomatic trees play an important role in the epidemiology of this disease, and avocado nurseries need to be certified to ensure they provide pathogen-free avocado material. Although there is no cure for infected trees, sanitation practices may have a significant impact on avoiding the spread of this pathogen.

Expresión diferencial de genes de defensa en aguacate en respuesta a la infección del viroide de la mancha de sol del aguacate

<p>En aguacate se ha demostrado la participación de proteínas relacionadas con patogénesis en la defensa contra algunos de sus patógenos más importantes. Sin embargo, en la enfermedad de la mancha de sol del aguacate causada por el <em>Avocado sunblotch viroid</em> (ASBVd) aún quedan algunos componentes de la respuesta de defensa por aclarar. En particular, la infección por ASBVd puede ser asintomática o causar síntomas, lo cual genera incógnitas sobre el mecanismo de defensa de la planta. En esta investigación se analiza la expresión de genes codificantes de proteínas de defensa, <em>PaNPR1</em>, <em>EREBP</em>, <em>PR-5</em> y <em>PR-6</em> en hojas y frutos de aguacate infectado por ASBVd, en condición sintomática y asintomática. El análisis de datos muestra que la infección de ASBVd modifica la expresión de los genes <em>PR-5 </em>y <em>PR-6</em>, mientras que la de <em>PaNPR1 </em>y <em>EREBP </em>no se modifica. Se encontraron diferencias en la expresión en frutos, siendo notable con <em>PR-5 </em>en frutos de árboles asintomáticos; a su vez, <em>PR-6</em> se expresa más en frutos infectados pero sin diferencias entre sintomático y asintomático. La expresión coordinada de ambos genes en frutos sugiere la activación de un mecanismo sinérgico de respuesta de defensa a la infección de ASBVd.</p>