Final Report Summary - VIRUSES AND APHIDS (Subversion of Insect Resistance: A Novel Role for Plant Viral Silencing Suppressor)
Project context and objectives
Aphids are important plant pests and act as vectors for the majority of plant-infecting viruses. The cucumber mosaic virus (CMV), an important aphid-transmitted pathogen, encodes the 2b counter-defence protein, which subverts plant antiviral responses, salicylic acid (SA)-mediated defence and small RNA-based RNA silencing. We found that infection of Arabidopsis (Arabidopsis thaliana) by CMV, but not CMVΔ 2b (a virus mutant lacking the 2b gene and so is unable to make the 2b protein), induced resistance to the green peach aphid (Myzus persicae). Conversely, infection with CMV but not CMVΔ 2b enhanced the resistance of tobacco (Nicotiana tabacum) to M. persicae (Westwood, Du et al, paper in preparation; Ziebell et al., 2011 Scientific Reports 1: 187).
To understand CMV-induced aphid resistance, we used NASC ATH1 Arabidopsis and tobacco DNA microarrays. We tested the effects of the 2b protein on plant gene expression during infection with CMV versus CMVΔ2b. In A. thaliana 920 and 275 genes were significantly altered by CMV and CMVΔ2b, respectively. Differential regulation of 616 genes by CMV compared with CMVΔ 2b was observed. Gene ontology analyses of up-regulated genes by CMV, using MapMan software, showed that the most represented biological processes are defence and stress responses. Up-regulated defence genes included pathogenesis-related gene 1 (PR1), PR2 (beta-glucanase 2/BGL2), PR5, phytoalexin deficient 3 (PAD3), and PAD4, which all are associated with signalling via the pathway regulated by salicylic acid - an important defensive hormone in plants. However, CMVΔ 2b did not efficiently activate any defence-related biological process. In tobacco, CMV also up-regulated many SA-responsive genes, including PR1, BGL2 and PR5. Again, this was due to CMV-induced increases in SA biosynthesis. Although CMV?2b markedly affected about 160 genes in tobacco, it really did not induce SA and JA signalling, based on currently limited gene ontology annotations derived from Arabidopsis data (Du, Murphy et al. paper in preparation).
Importantly, ATH1 microarray data showed that CMV up-regulated genes (PAD4, CYP81F2, MYB44, MYB51) associated with Arabidopsis resistance against aphids. PAD4 is a regulator in the feedback loop regulating SA biosynthesis. Experiments with Arabidopsis mutants pad4 and sid2 (deficient in SA biosynthesis) indicated that CMV-induced resistance to aphids is SA-independent. MYB44, MYB51 and CYP81F2 all are involved in glucosinolate metabolite. HPLC analysis of glucosinolates showed that the anti-aphid compound, 4-methoxy-indolyl-3-methyl glucosinolate (4MI3M) was induced in Fny-CMV-infected plants, but not in Fny-CMVΔ2b-infected plants, proposing that CMV-induced resistance to aphid is probably related with the increased accumulation of 4MI3M via the up-regulation of genes CYP81F2 and MYB51 (Westwood, Du et al. paper in preparation).
CMV strains are divided into three subgroups (IA, IB and II). Our previous work showed that the 2b protein encoded by the Fny-CMV strain (from subgroup IA: the strain used for work described above) interferes with RNA silencing pathways mediated by both MIRNA and SIRNA. However, 2b proteins of Q-CMV or LS-CMV (subgroup II) only interfered with RNA silencing pathways mediated by SIRNAs. We used 2b genes from all three subgroups to assess the relative importance of the pathways on plant resistance to aphids. We tested for aphid resistance in Arabidopsis infected with Fny-CMV, LS-CMV, and FCb72b-CMV (in which the Fny-CMV 2b gene was replaced with that of subgroup IB strain Cb7-CMV). Interestingly, both Fny-CMV and FCb72b-CMV induced aphid resistance but not LS-CMV. Experiments with 2b-transgenic Arabidopsis indicated that Fny2b- and Cb72b-transgenic lines had increasing host resistance to aphids, but LS2b-transgenic plants not. Furthermore, investigation with mutants in the MIRNA pathways (ago1 and dcl1) showed that aphids had a significantly lower colony size and an average growth rate compared to wild-type plants, but there is no difference observed between dcl2/3/4 (compromised in the SIRNA pathway) and wild-type plants. Thus, the MIRNA pathway negatively regulates Arabidopsis resistance to aphids, and 2b-induced resistance to aphids appears to involve the disruption of the MIRNA pathway. To find out which MIRNA(s) are involved in CMV/2b-induced resistance to aphids, we had successfully established CMV-derived vector to block MIRNA activity ('MIRNA sponge' approach). We are making an effort to test specific MIRNA(s) molecule or family using the CMV vector.
Project results
The project objectives were achieved and novel findings generated were notably: the CMV infection induced an accumulation of the anti-aphid compound 4MI3M in Arabidopsis; the MIRNA pathway negatively regulates Arabidopsis resistance against aphids; and independent of CMV infection, it enhanced Arabidopsis resistance to aphids via the MIRNA pathway.
The socio-economic impact in China has been affected as the yield of important crops have significantly decreased due to, in a large part, a virus infection. Aphids are the vector of CMV, an economically important virus that prevails in China and Europe. The Chinese government is focusing resources on developing agriculture and investing more funding in agricultural research. Using data from this project, Zhiyou Du has been awarded funding from the China National Natural Science Foundation to further investigate CMV-induced resistance to aphids with the collaboration of Dr John Carr. Thus, we are using the data from this project to produce more and long-term collaboration between Europe and China.
Contacts
Dr Zhiyou Du, Institute of Bioengineering, Zhejiang Sci-Tech University, Hangzhou, China 310018 (contact duzhiyou@yahoo.com.cn for information)
Dr John P. Carr, Department of Plant Sciences, University of Cambridge, Downing St., Cambridge CB2 3EA, UK (contact jpc1005@cam.ac.uk for information).