Phytoplasma effector SAP05 induces witches' broom in Arabidopsis [John Innes Centre]

The results of studies in a plant model organism suggest that a newly discovered mechanism used by parasitic bacteria to slow down plant aging may point to a novel approach to protecting disease-threatened food crops. The research, headed by scientists at the John Innes Centre, identified a molecule called SAP05, which Phytoplasma bacteria produce and use to hijack plant development. When inside the infected plant, the protein causes the breakdown of key growth regulators, triggering abnormal growth.

Saskia Hogenhout, PhD, professor, John Innes Centre and corresponding author of the team’s published report in Cell, said, “Phytoplasmas are a spectacular example of how the reach of genes can extend beyond the organisms to impact surrounding environments. Our findings cast new light on a molecular mechanism behind this extended phenotype in a way that could help solve a major problem for food production. We highlight a promising strategy for engineering plants to achieve a level of durable resistance of crops to phytoplasmas.” Hogenhout and colleagues described their findings in a paper titled, “Parasitic modulation of host development by ubiquitin-independent protein degradation.”

Parasites manipulate the organisms they live off to suit their needs, sometimes in drastic ways. Some plants infected by particular types of parasites, for example, undergo such extensive changes that they are described as “zombies.” The plants may stop reproducing and serve only as a habitat and host for the parasitic pathogens. But until now, there’s been little understanding of how this happens on a molecular and mechanistic level.

Phytoplasma belong to a group of bacteria that are notorious for their ability to reprogram the development of their host plants. Phytoplasmas are strict obligate parasites, and have a dual host cycle that alternates between plants and insects. This group of bacteria is often responsible for the “witches’ brooms” seen in trees, which occur when an excessive number of branches grow close together. The bushy outgrowths are the result of the plant being stuck in a vegetative “zombie” state, unable to reproduce, and therefore progress to a “forever young” status.

As the authors explained, “Phytoplasmas infect most vascular plant species and often induce massive changes in plant architecture, such as excessive proliferation of shoots and branches (witches’ broom) and retrograde development of flowers into leaf-like organs (phyllody) … Phytoplasma-infected plants have been referred to as ‘‘zombie plants’’ because they exhibit extensive architectural changes, stop reproducing, and appear to serve solely as habitats for the phytoplasma pathogens and their insect vectors.” Phytoplasma bacteria can also cause devastating crop disease, such as Aster Yellows, which causes significant yield losses in both grain and leaf crops like lettuce, carrots, and cereals.

The newly reported findings have shown how the bacterial protein SAP05 manipulates plants by taking advantage of some of the host’s own molecular machinery. This machinery, called the proteasome, usually breaks down proteins that are no longer needed inside plant cells. SAP05 hijacks this process, causing plant proteins that are important in regulating growth and development, to effectively be thrown in a molecular recycling center. Without these proteins, the plant’s development is reprogrammed to favor the bacteria, triggering the growth of multiple vegetative shoots and tissues and putting the pause on plant aging.

Through genetic and biochemical experiments on the model plant Arabidopsis thaliana, the team uncovered in detail the role of SAP05. They found that SAP05 binds directly to the plant developmental proteins and also interacts with the 26S proteasome subunit RPN10. “… we discovered that a phytoplasma effector, SAP05, binds and mediates degradation of multiple members of two distinct transcription factor families, the SPL family and the GATA family, leading to delayed plant aging and simultaneous proliferation of vegetative tissue and shoots,” the researchers noted. SPL transcription factors play a conserved role in controlling developmental phase transitions of vascular plants, whereas GATA transcription factors regulate plant organ development, timing of flowering, and branching patterns in dicot and monocot plants, the researchers further explained.

This direct binding is a newly discovered way to degrade proteins. Usually, proteins that are degraded by the proteasome are tagged with a molecule called ubiquitin beforehand, but this doesn’t happen in the mechanism resulting from phytoplasma infection. “SAP05 mediates degradation through a ubiquitination-independent mechanism by co-opting the 26S ubiquitin receptor RPN10, which is highly conserved across eukaryotes,” the scientists commented. “RPN10 locates within the 19S regulatory particle of the 26S proteasome and serves as one of the main ubiquitin receptors recruiting ubiquitinated proteins for proteasomal degradation.”

The plant developmental proteins that are targeted by SAP05 are similar to proteins also found in animals. The team was curious to see if SAP05 therefore also affects the insects that carry the bacteria plant to plant. They found that the structures of these host proteins in animals differ enough that they do not interact with SAP05, and so the protein does not affect the insects. “RPN10 is highly conserved among eukaryotes, but SAP05 does not bind insect vector RPN10,” the team further stated.

The study results allowed the team to pinpoint just two amino acids in the proteasome unit that are needed to interact with SAP05. Their research showed that if the plant proteins are switched to have the two amino acids found in the insect protein instead, they are no longer degraded by SAP05, preventing the abnormal growth of witches’ broom. “The SAP05-binding specificity to RPN10 can be dependent on just two amino acids that, fascinatingly, are one of the only few sequence differences between plant and human/animal RPN10 vWA domains,” the investigators explained. “A two-amino-acid substitution within plant RPN10 generates a functional variant that is resistant to SAP05 activities.”

This finding offers the possibility of tweaking just these two amino acids in crops—for example, by using gene-editing technologies such as CRISPR-Cas—could provide durable resilience to phytoplasmas and the effects of SAP05. “Our model describes a mechanistic framework for how obligate parasites can induce complex and substantial phenotypic changes in their hosts in ways that favor their transmission to other trophic levels,” the team concluded. “Hence, introduction of single-nucleotide changes in RPN10 genes (for example, by CRISPR-Cas technologies …) is a promising strategy to achieve durable resistance of crops to phytoplasmas.”

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