Sangsuwan, Parisatcha
(2020)
The role of phytoplasma effectors in plant development.
PhD thesis, University of Nottingham.
Abstract
One of the problems that can cause yield losses in crops is infection with phytoplasmas. In monocots, phytoplasmas cause serious problems in economic plants such as rice yellow dwarf (RYD), napier grass stunt disease (NGSD), sugarcane white leaf (SCWL) and sugarcane grassy shoot (SCGS). This research aims to identify phytoplasma effector proteins causing these symptoms, which is potentially of benefit for future studies to prevent host plants from getting the symptoms associated with phytoplasma effector proteins. Furthermore, phytoplasmas of gramineous plants were classified using the MLST technique into new groups or subgroups, particularly focusing on sugarcane diseases, because the 16Sr gene cannot classify closely related strains.
First of all, ten genes were used to classify the phytoplasmas of gramineous plants such as NGSD, SCWL, SCGS and Bermuda grass white leaf (BGWL) and the results showed that the lpd, gro, gryB, ser and sec gene primers classified the SCWL from the SCGS based on geographic origin. Interestingly, the result showed that the NGSD and BGWL phytoplasmas were quite distinct from those of sugarcane. These results support that the BGWL are correctly classified into the 16Sr XIV group whilst the NGSD should be classified into a new group. Primers based on the asp, 50S, secA, EF2 and EF7 genes were less useful for classification, especially the EF2 and EF7 primers, which are for putative effector genes, presumably because these genes require conserved regions to interact with plant hosts whilst pep primers could not amplify any samples. For future studies, the MLST analysis should include the effector genes and analysis of environmental factors such as soil type, soil moisture and soil nutrients should also be undertaken to help identify the causes of the different sugarcane symptoms.
Secondly, the NGSD genomic sequence contigs were analysed for secretion signals using bioinformatics and the results identified fourteen proteins that had the signal peptide sequences required to make them putative effectors. Seven of the putative effector genes from NGSD and two from SCWL were cloned and transformed into Arabidopsis, and for controls, SAP11 genes (which is a known effector) were cloned from a range of other phytoplasmas held in the University of Nottingham collection. The results identified five SAP11 genes from the 16SrI Blackcurrant reversion disease (BCRD) phytoplasma, 16SrI Strawberry green petal (SGP), 16SrII Vinca coconut phytoplasma (VCP), 16SrII Cotton phyllody (CoP) and 16SrX Apple proliferation (AP_SQ). Interestingly, the SAP11AP_SQ sequence was different from that in the NCBI database for the whole genome sequenced strain of Apple proliferation, indicating difference within 16Sr groups.
In the screening step, symptoms and gene expression using absolute quantitative PCR were analysed in the 2nd generation of transformed Arabidopsis. The transformed plants showed various phenotypes; phyllotaxis defects for SAP11AP_SQ, SAP11BCRD, SAP11CoP and SAP11VCP; branching for SAP11AP_SQ, SAP11CoP, SAP11SGP, EF2NGSD, EF7NGSD, EF2SCWL and EF7SCWL. Furthermore, SAP11AP_SQ and SAP11VCP transformed plants were shorter than wild type and vector only plants. Moreover, SAP11AP_SQ transformed plants produced clustered cauline leaves on the primary inflorescence and the SAP11AP_SQ did not produce flowers, whilst the SAP11VCP transformed flowers were smaller than wild type and vector only, although they were still fertile. These phenotypes result from SAP11 interacting with plant genes such as BRC1 and PME5 which control branching and phyllotaxis patterns respectively, BP which controls internode elongation and PNF and PNY which maintains inflorescence architecture and floral meristem function. According to the transformation results, the effector genes from NGSD and SCWL were successfully transformed into Arabidopsis; however, the transformed plants did not show any symptoms related to stunting or white leaf. Therefore, transformation into monocot models such as rice, barley or Brachypodium needs to be undertaken to observe the symptoms that might be specific to the host range. However, attempts were also made to undertake transformation in the monocot Brachypodium BD21 for symptom observation, but no results were obtained due to the time taken to obtain transformed plants and the closure of labs due to Covid-19. So, future studies will repeat transformation into monocots and observe the symptoms including plant gene expression levels.
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