Functional characterization of secreted proteins on the SAP11 region of the Aster Yellows phytoplasma strain Witches' Broom genome

Phytoplasmas are bacterial plant pathogens that have been associated with massive yield losses of crops worldwide. They are particularly devastating for high-value production crops, such as blackberry, apple and pear productions in the United Kingdom; grape vines in southern Europe; and palm trees in Africa, the Caribbean Islands, Florida and Mexico. They are also prevalent in wild plant species, and are associated with losses of native plants unique to some areas. Phytoplasmas are transmitted from plant to plant by phloem-feeding insects in which they are predominantly dependent on their plant and insect hosts for dispersal in nature. They have the unique ability to invade and replicate in their plant and insect hosts. Furthermore, some phytoplasmas, including Aster Yellows phytoplasma strain Witches' broom (AY-WB), manipulate plant species to become better hosts for phloem-feeding insects, including insects that normally do not survive on these species. Thus, apparently, phytoplasmas can interfere with plant defense responses to insect attack. Phytoplasmas also interfere with plant developmental processes as they can induce dramatic symptoms in plants such as witches' broom (growth of a dense mass of shoots from a single point) and phyllody (retrograde metamorphosis of the floral organs to the condition of leaves). Genome sequencing and subsequent bioinformatics analyses have revealed that phytoplasmas produce a range of effector proteins of which some target plant cell nuclei and can perturb plant processes. In this proposal, we focus on the characterization of some of the effector proteins of AY-WB. We will determine whether these effector proteins are involved in symptom development and in increased attractiveness of plants to insect vectors.

Phytoplasmas are bacterial plant pathogens that predominantly depend on sap-sucking hemipteran insects, such as leafhoppers, for their dispersal in nature. They have the unique capability to replicate in their plant hosts and insect vectors, and are known to manipulate both for increasing infection and dispersal efficiencies. Many phytoplasmas have a broad host range. The phytoplasma studied in this proposal, Aster Yellows phytoplasma strain Witches' Broom (AY-WB), can infect China aster, lettuce, Nicotiana benthamiana, tomato, Arabidopsis thaliana and maize, and is also capable of infecting insects, such as its leafhopper vector Macrosteles quadrilineatus. Furthermore, AY-WB-infected plants are better hosts for potential insect vectors that normally do not survive on some of these plant species. In recent years, we made considerable progress in understanding the molecular basis of phytoplasma interactions with plants and insects. Mining of the complete AY-WB genome sequence has resulted in the identification of 56 secreted proteins (SAPs) that are potential effector proteins. Unlike Gram-negative bacterial plant pathogens, phytoplasmas do not require a type III secretion system. These bacteria use the Sec-dependent pathway for introducing effector proteins into host cells. Because phytoplasmas locate mostly intracellularly, they deliver secreted proteins directly into the host cell cytoplasm. One of the effector proteins, SAP11, targets the nuclei of plant cells and changes plant gene transcription profiles. SAP11 can unload from the phloem to target young developing plant tissues, consistent with the witches' broom symptoms that AY-WB induces in developing shoots. The SAP11 gene is located on a mobile region (the SAP11 region) reminiscent of a pathogenicity island that contains genes for 5 other effector proteins. In this proposal, we will focus further research on the functional analysis of the 6 effector proteins of the SAP11 region.