Pathology of Hop Latent Viroid in Cannabis sativa

We have done some work to smoke out a potential mechanism of HpLVd pathology in Cannabis. To help visualize this, we built Viropedia.net. This tool maps the variants found in your HpLVd genome to the Jamaican Lion transcriptome so you can see the regions in the Viroid that have homology to the cannabis mRNAs (click on the Zoomable link and drag the molecule around, you'll thank us later:). This is important once you recognize the pathology of other viroids is RNA interference based and its likely the same mode with HpLVd. What is RNA interference? Short sequences in the HpLVd genomes get diced up by RNases called DICER and then interfere with the expression of genes they have the most sequence homology with in Cannabis. So, how much homology is there?

Spoiler alert... There is a lot. We find 25 mRNAs in C.sativa that have 17-25mer homology to the various HpLVd genomes. COG7 is the most conserved homology found in 69% of the published HpLVd genomes. Kannapedia.net has many cultivars with variants in these targets suggesting polymorphisms in certain cultivars may limit the RNA interference of HpLVd. This enables a path for selective breeding for cultivars more tolerant of HpLVd.

HpLVd is believed to have a replication error rate of 1 error in 500 nucleotide based on estimated measured in its closest cousins in Pospiviroidae. This results in Multiple Sequence Alignments seen at the header of this post where only 2 bases in the genome are invariant across all 162 NCBI entries. One of these invariant bases is the 3 prime end of the COG7 homology to C.sativa.

This RNA polymerase error results in a diversity of secondary structure. Below is an example from RNAfold of a viroid that only differs by 2 SNPs, yet results in completely different secondary structure. Of note is the Quadruplex G structure we found in the CCR. These are important features as they recruit lots of plant proteins and are likely key to the catalytic activity of the viroid.

Viropedia.net has these homologies decorated for each variant in NCBI.

While short homologies are interesting, they don't always translate into RNA interference. We designed a qPCR assay to monitor COG7 expression in infected and non-infected plants. Preliminarily, we see a change (delta CT) but the plants in this study were not clones of each other and this work needs expansion into more cultivars.

We are actively looking to work with growers and labs to further understand this pathway as we believe it is the only path off the qPCR treadmill... ie, we need to start breeding for plants that are more tolerant of this viroid. The viroid has other plant reservoirs and reservoirs that may also include fungi so complete elimination is unlikely and hasn't occurred in Hop despite the fields many more years experience at attempting this. The first Hop Latent Viroid was published in 1988. It is still around.

What is COG7? It is a gene in Cannabis sativa that is expressed in most tissues and is required for Shoot Apical Meristem growth. The knock outs found in Arabidopsis are interesting.

But COG7 isn't the only story. There are homologies to 24 other mRNAs described in our Preprint. Most have literature support in that other viroid studies have come to similar conclusions in tomato. Callose synthase, EXLA1 and even PCMP-E102 are prime suspects but these hits are only found in a minority of HpLVd genomes to date.

You can read more about this in our latest PrePrint on the topic.

We are actively sequencing growers HpLVd genomes and Cannabis genomes to sort this out. Contact us if you want to build a long term plan that can greatly augment the qPCR screening currently being deployed.