Skip to main content

A characterization of the effectors of a plant pathogen

Final Report Summary - AY-WB EFFECTORS (A characterisation of the effectors of a plant pathogen)

Background information

Phytoplasmas are insect-transmitted pathogens of many plants. These bacteria are members of the class Mollicutes and are related to the Mycoplasmas that cause disease in humans and other mammals. Phytoplasmas are transmitted to plants by insects that feed from the phloem (or sap) of the plant, including leafhoppers, planthoppers, and psyllids (Sugio et al., 2011). Infected plants exhibit many symptoms that reflect alterations of development, such as the growth of leaf-like flowers (a process referred to as phyllody) and witches' broom (enhanced outgrowth of stems from the plant). Once infected, plants cannot be cured of the bacteria, and thus current control measures typically rely upon the application of environmentally damaging pesticides as a means of reducing insect vector populations.

At a socio-economic level, phytoplasmas adversely affect global food production and security. These bacteria cause serious disease in many agriculturally important food crops in Europe and the Americas, including grapevine, apple, potato, peanut, alfalfa, stone fruit, carrot, lettuce, and maize. Phytoplasmas are also responsible for devastating outbreaks of coconut lethal yellowing diseases (CLYD) of Africa and the Caribbean, resulting in the impoverishment of local farmers. According to the Food and Agriculture Organisation of the United Nations (FAO), CLYD has resulted in the loss of approximately 40 % coconut palms in Tanzania, and has impoverished 20 % of rural communities in Ghana that depend upon coconuts as a primary food source. The productivity of European crops has also been adversely affected by phytoplasmas, with grape vineyards (loss of 10 to 80 % total harvest) and apple orchards (10 to 70 %) suffering significant economic losses.

Despite the economic importance of these organisms, little is known about how phytoplasmas regulate interaction with respective hosts. This project seeks to elucidate the mechanism(s) by which phytoplasmas infect plants and cause disease. Knowledge generated from this research will help in producing phytoplasma-resistant crops and thus will contribute towards enhancing global food security.

The hypothesis of the research project is that phytoplasmas employ effectors (or virulence proteins) that actively modulate the development of infected plants.

The objectives of this project are twofold:
(i) to determine how the effector genes are regulated; and
(ii) to functionally characterise putative phytoplasma effectors to determine whether these proteins modulate plant phenotype.

Overview of results

The genome of Aster Yellows phytoplasma strain Witches' Broom (AY-WB) is predicted to encode 56 effector genes (Bai et al., 2009). As a means of examining the regulation of these genes, we determined their expression levels in insect- and plant-colonising AY-WB. Approximately, one half of the putative effector genes were upregulated in an insect host (the aster leafhopper Macrosteles quadrilineatus) whereas the other half were highly expressed in Arabidopsis. These data strongly suggest that phytoplasmas such as AY-WB employ a host-specific profile of effector proteins and highlights potential effectors that are likely to be relevant to the infection of crops (i.e. encoded by genes that are up-regulated in planta).

We selected 6 putative AY-WB effectors (SAP27, SAP41, SAP43, SAP54, SAP61, and SAP67) to assess whether these proteins have the ability modulate plant development. Of these, we determined that expression of SAP54 in transgenic Arabidopsis induces the growth of leaf-like flowers (phyllody) with green petals (virescence) that closely resemble flowers produced from phytoplasma-infected plants. We wished to further characterise SAP54, and thus employed a yeast two-hybrid strategy to identify plant proteins that are recognised and bound by the bacterial effector. Our two-hybrid screen was performed using a library of Arabidopsis seedling complementary deoxyribonucleic acid (cDNA), and revealed that SAP54 interacts with members of the MADS-domain transcription factor family. We extended our analyses to include all (106) MADS-domain proteins encoded in the Arabidopsis genome, and determined that SAP54 interacts with a subset of Type II canonical MADS-domain proteins, including SEP3, AP1, and SOC1. MADS-domain proteins represent a broadly conserved group of regulatory proteins that play key roles in regulating the timing of flowering and the formation of floral organs (for example, the growth of sepals and petals) (as reviewed in Smaczniak et al., 2012).

We hypothesised that the interaction between SAP54 and MADS-domain proteins was relevant to the formation of leaf-like flowers in phytoplasma-infected plants, and further characterised the association between these proteins. We observed that SAP54 induces the degradation of the MADS-domain proteins when expressed in planta, and further, that this degradation process involves the host ubiquitin-26S proteasome system. Thus, it would seem that the formation of green, leafy, flowers from phytoplasma-infected or SAP54-expressing plants is due to the destabilisation of key regulatory plant transcription factors by SAP54.

Our yeast two-hybrid screen also revealed that SAP54 interacts with Arabidopsis proteins RAD23C and RAD23D. RAD23C-D function as shuttling proteins that transport poly-ubiquitinated substrates to the ubiquitin-26S proteasome system for degradation (Farmer et al., 2010). We have demonstrated that SAP54 interacts with RAD23C-D in Arabidopsis via co-immunoprecipitation experiments. Critically, we observe that rad23CD double mutants produce normal flowers when transformed with the green fluorescent protein (GFP)-SAP54 whereas transformation of GFP-SAP54 into Col0 results in the production of green, leaf-like flowers. This observation indicates that RAD23C-D are essential for SAP54-mediated phenotypes of phyllody and virescence. We have proposed a model by which phytoplasma such as AY-WB induces symptoms of phyllody in infected plants.

Conclusions and socio-economic impacts

This research project has led to the identification of an entirely novel effector protein. SAP54 induces phyllody and virescence in plants by destabilising MADS-box proteins, and requires RAD23C-D as helper proteins to facilitate this process. The potential impact of this discovery is significant. Phytoplasmas infect many important agricultural crops (such as grapevine, apples, pears, apricots, plums, and peaches) and these pathogens reduce both the quality and quantity of fruit harvests by interfering with flowering and fruiting of infected orchards. Our research has identified SAP54 as a phytoplasma effector that may play an important role in this process, and has identified the mechanism by which SAP54 acts (i.e. destabilising MADS-box transcription factors). This knowledge will greatly aid in the generation of phytoplasma-resistant crops, and will thus be of direct benefit to European (and world) farmers and contributes towards improving global food security.

References:

(1) Sugio et al., 2011. Annual Review of Phytopathology 49:175-195.
(2) Bai et al., 2009. Molecular Plant-Microbe Interactions 22:18-30.
(3) Smaczniak et al., 2012. Development 139:3081-3098.
(4) Farmer et al., 2010. Plant Cell 22:124-142.

Related documents