Reactive oxygen species-related genes participate in resistance to cucumber green mottle mosaic virus infection regulated by boron in Nicotiana benthamiana and watermelon.

Cucumber green mottle mosaic virus (CGMMV) infection causes acidification and rot of watermelon flesh, resulting in serious economic losses. It is widely reported the interaction relationship between boron and reactive oxygen species (ROS) in regulating normal growth and disease resistance in plants. Our previous results demonstrated that exogenous boron could improve watermelon resistance to CGMMV infection. However, the roles of ROS-related genes regulated by boron in resistance to CGMMV infection are unclear. Here, we demonstrated that CGMMV symptoms were alleviated, and viral accumulations were decreased by boron application in Nicotiana benthamiana, indicating that boron contributed to inhibiting CGMMV infection. Meanwhile, we found that a number of differentially expressed genes (DEGs) associated with inositol biosynthesis, ethylene synthesis, Ca2+ signaling transduction and ROS scavenging system were up-regulated, while many DEGs involved in ABA catabolism, GA signal transduction and ascorbic acid metabolism were down-regulated by boron application under CGMMV infection. Additionally, we individually silenced nine ROS-related genes to explore their anti-CGMMV roles using a tobacco rattle virus (TRV) vector. The results showed that NbCat1, NbGME1, NbGGP and NbPrx Q were required for CGMMV infection, while NbGST and NbIPS played roles in resistance to CGMMV infection. The similar results were obtained in watermelon by silencing of ClCat, ClPrx or ClGST expression using a pV190 vector. This study proposed a new strategy for improving plant resistance to CGMMV infection by boron-regulated ROS pathway and provided several target genes for watermelon disease resistance breeding.

Suppression of Cucumber Green Mottle Mosaic Virus Infection by Boron Application: From the Perspective of Nutrient Elements and Carbohydrates.

Cucumber green mottle mosaic virus (CGMMV) infection causes "blood flesh" symptoms in watermelon fruits, which severely reduces yield and edibleness. However, the growth of watermelon fruits is strongly associated with boron (B), a trace element for improving fruit quality. In this study, B-gradient hydroponic experiments (B concentration: 0, 2.86, and 5.72 mg·L-1 H3BO3) and foliar-spray experiments (B concentration: 30 and 300 mg·L-1 H3BO3) were performed. We found that the B-supplement could inhibit CGMMV infection and especially relieve "blood flesh" symptoms in watermelon fruits. The nutrient element, soluble sugar, and cell wall polysaccharide contents and their metabolism- and transport-related gene expressions were determined in leaves and fruits of the watermelons in B-gradient hydroponic and foliar-spray experiments. We found that the accumulation and metabolism of nutrients and carbohydrates in cells were disrupted by CGMMV infection; however, the B-supplement could restore and maintain their homeostasis. Additionally, we uncovered that NIP5;1 and SWEET4, induced by B-application with CGMMV infection, could majorly contribute to the resistance to CGMMV infection by regulating nutrient elements and carbohydrate homeostasis. These results provided a novel insight into the molecular mechanism of B-mediated CGMMV suppression and an efficient method of B-application for the improvement of watermelon quality after CGMMV infection.

A novel strategy for improving watermelon resistance to cucumber green mottle mosaic virus by exogenous boron application.

The molecular mode controlling cucumber green mottle mosaic virus (CGMMV)-induced watermelon blood flesh disease (WBFD) is largely unknown. In this study, we have found that application of exogenous boron suppressed CGMMV infection in watermelon fruit and alleviated WBFD symptoms. Our transcriptome analysis showed that the most up-regulated differentially expressed genes (DEGs) were associated with polyamine and auxin biosynthesis, abscisic acid catabolism, defence-related pathways, cell wall modification, and energy and secondary metabolism, while the down-regulated DEGs were mostly involved in ethylene biosynthesis, cell wall catabolism, and plasma membrane functions. Our virus-induced gene silencing results showed that silencing of SPDS expression in watermelon resulted in a higher putrescine content and an inhibited CGMMV infection correlating with no WBFD symptoms. SBT and TUBB1 were also required for CGMMV infection. In contrast, silencing of XTH23 and PE/PEI7 (low-level lignin, cellulose and pectin) and ATPS1 (low-level glutathione) promoted CGMMV accumulation. Furthermore, RAP2-3, MYB6, WRKY12, H2A, and DnaJ11 are likely to participate in host antiviral resistance. In addition, a higher (spermidine + spermine):putrescine ratio, malondialdehyde content, and lactic acid content were responsible for fruit decay and acidification. Our results provide new knowledge on the roles of boron in watermelon resistance to CGMMV-induced WBFD. This new knowledge can be used to design better control methods for CGMMV in the field and to breed CGMMV resistant watermelon and other cucurbit crops.

Chimeric Tobamoviruses With Coat Protein Exchanges Modulate Symptom Expression and Defence Responses in Nicotiana tabacum.

In the pathogen infection and host defence equilibrium, plant viruses have evolved to efficiently replicate their genomes, to resist the attack from host defence responses and to avoid causing severe negative effect on growth and metabolism of the hosts. In this study, we generated chimeric tobacco mosaic virus (TMV) variants, in which the coat protein (CP) sequences were substituted with that of cucumber green mottle mosaic virus (CGMMV) or pepper mild mottle virus (PMMoV) to address the role of these in virus infection and host symptomology. The results showed that the chimeric viruses (TMV-CGCP or TMV-PMCP) induce stunting and necrotic symptoms in tobacco plants. We analyzed the transcriptomic changes in tobacco plants after infection of TMV and its chimeras using a high-throughput RNA sequencing approach and found that infection of the chimeric TMV induced significant up-regulation of host defence responsive genes together with salicylic (SA) or abscisic acid (ABA) responsive genes, but down-regulation of auxin (Aux) responsive genes. We further confirmed the increase in the levels of SA and ABA, together with the reduced levels of Aux after infection of chimeric TMV in tobacco plants. These data suggest novel roles of tobamovirus CP in induction of host symptoms and defence responses.

iTRAQ-Based Proteomic Analysis of Watermelon Fruits in Response to Cucumber green mottle mosaic virus Infection.

Cucumber green mottle mosaic virus (CGMMV) is an important viral pathogen on cucurbit plants worldwide, which can cause severe fruit decay symptoms on infected watermelon (usually called "watermelon blood flesh"). However, the molecular mechanism of this disease has not been well understood. In this study, we employed the isobaric tags for relative and absolute quantitation (iTRAQ) technique to analyze the proteomic profiles of watermelon fruits in response to CGMMV infection. A total of 595 differentially accumulated proteins (DAPs) were identified, of which 404 were upregulated and 191 were downregulated. Functional annotation analysis showed that these DAPs were mainly involved in photosynthesis, carbohydrate metabolism, secondary metabolite biosynthesis, plant-pathogen interaction, and protein synthesis and turnover. The accumulation levels of several proteins related to chlorophyll metabolism, pyruvate metabolism, TCA cycle, heat shock proteins, thioredoxins, ribosomal proteins, translation initiation factors, and elongation factors were strongly affected by CGMMV infection. Furthermore, a correlation analysis was performed between CGMMV-responsive proteome and transcriptome data of watermelon fruits obtained in our previous study, which could contribute to comprehensively elucidating the molecular mechanism of "watermelon blood flesh". To confirm the iTRAQ-based proteome data, the corresponding transcripts of ten DAPs were validated by determining their abundance via quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). These results could provide a scientific basis for in-depth understanding of the pathogenic mechanisms underlying CGMMV-induced "watermelon blood flesh", and lay the foundation for further functional exploration and verification of related genes and proteins.

Development of a multiplex RT-PCR assay for simultaneous detection of Cucumber green mottle mosaic virus and Acidovorax citrulli in watermelon.

Watermelon (Citrullus lanatus Thunb.) is considered as a popular and nutritious fruit crop worldwide. Watermelon blood flesh disease caused by Cucumber green mottle mosaic virus (CGMMV) and bacterial fruit blotch caused by Acidovorax citrulli, are two major quarantine diseases of watermelon and result in considerable losses to global watermelon production. In this study, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) method was developed for simultaneous detection of CGMMV and A. citrulli in both watermelon leaves and seeds. Two pairs of specific primers were designed based on the conserved sequences of the genomic RNA of CGMMV and the internal transcribed spacer of A. citrulli, respectively. Transcriptional elongation factor-1α from watermelon was added as an internal reference gene to prevent false negatives. No cross-reactivity was detected with other viral or bacterial pathogens infecting watermelon. Moreover, the multiplex RT-PCR showed high sensitivity and could simultaneously detect CGMMV and A. citrulli as little as 102 copies of plasmid DNA. This method was successfully applied to test field-collected watermelon leaves and stored seeds of cucurbitaceous crops. These results suggested that the developed multiplex RT-PCR technique is a rapid, efficient, and sensitive method for simultaneous detection of CGMMV and A. citrulli, providing technical support for monitoring, predicting, and preventing these two quarantine diseases. To our knowledge, this is the first report on simultaneous detection of a virus and a bacterium by multiplex RT-PCR in watermelon.