Viroids are naked

One of the most intriguing aspects of viroid research, for me, is their simplicity. And yet, despite their simplicity, we still do not fully understand them.

I am Jernej Jakše, a professor of genetics at the University of Ljubljana. I earned my PhD 21 years ago with a thesis focused on microsatellite markers in hops, known host plant of viroids.
 
Hop plants are currently known to host four different viroids: hop latent viroid (HLVd), hop stunt viroid (HSVd), apple fruit crinkle viroid (AFCVd), and citrus bark cracking viroid (CBCVd). HSVd was the first viroid discovered in hops, but it remained restricted to Japan for a long period (Sasaki and Shikata, 1977). HLVd was the second viroid identified in hop plants, with evidence emerging in 1987 (Pallas et al., 1987) and its sequence determined in 1988 (Puchta et al., 1988).
 
Although HLVd infections are asymptomatic—and nearly all commercially grown hop plants are infected—it was initially considered harmless to hop production. Nevertheless, diagnostic techniques were developed to detect HLVd in hops, supporting efforts to obtain viroid-free plants.
 
As a Master's student, I first encountered reverse PAGE electrophoresis for detecting HLVd in our laboratory. At the time, a PhD student was refining this detection approach, and I vividly remember the challenges, such as cracked glass plates during the denaturation process.
 
Later, in 2003, I joined a project with Dr. Sebastjan Radišek from the Slovenian Institute of Hop Research and Brewery in Žalec. Together, we developed a RT-PCR method and a non-radioactive detection method for HLVd (see Fig. 1 and Fig. 2). For roughly a decade, I stepped away from active viroid research, only to return when Sebastjan and I tackled the mystery of stunted hop plants in Slovenian fields. This investigation led to the discovery of CBCVd infection (Jakse et al., 2015). Since then, viroid research has once again become a part of my work. I've also built fruitful collaborations with various researchers and groups across Europe.
 
These collaborations culminated in the successful ViroidDoc network training, which I am excited to participate in. One of the most intriguing aspects of viroid research, for me, is their simplicity. Viroids are naked, circular RNAs, ranging from 250 to 400 base pairs, with no coding potential. And yet, despite their simplicity, we still do not fully understand them.
 
 
Jernej Jakše, PhD, is a full professor at the University of Ljubljana – Biotechnology, Department of Agronomy, Chair of Genetics, Biotechnology, Statistics and Plant Breeding.

Figure 1: Detection of three different hop total nucleic acid (TNA) samples using dot-blot hybridization. Samples of 5, 1, or 0.1 μl, isolated with AMES or CTAB extraction buffer, were applied to the membrane. Both extraction buffers produced signals of approximately equal intensity, but better results were obtained in RT-PCR with CTAB samples.
 
Figure 2: RT-PCR results of 10 hop samples isolated with AMES and CTAB extraction buffers. The expected HLVd fragment, 250 bp in length, was amplified in all 10 CTAB samples. Strong inhibition was observed with the AMES extraction buffer, as the HLVd fragment was amplified in only two samples (7 and 21); a – sample without TNA (negative control), b – control RNA amplification (323 bp, a component of the Promega kit).

1. JAKSE, J., RADISEK, S., POKORN, T., MATOUSEK, J. & JAVORNIK, B. 2015. Deep‐sequencing revealed Citrus bark cracking viroid (CBCVd) as a highly aggressive pathogen on hop. Plant Pathology.
2. PALLAS, V., NAVARRO, A. & FLORES, R. 1987. ISOLATION OF A VIROID-LIKE RNA FROM HOP DIFFERENT FROM HOP STUNT VIROID. Journal of General Virology, 68, 3201-3205.
3. PUCHTA, H., RAMM, K. & SANGER, H. L. 1988. The molecular-structure of hop latent viroid (HLV), a new viroid occurring worldwide in hops. Nucleic Acids Research, 16, 4197-4216.
4. SASAKI, M. & SHIKATA, E. 1977. On some properties of hop stunt disease agent, a viroid. Proceedings of the Japan Academy, Series B. Physical and Biological Sciences, 53, 109-112.