Abstract

   Cucumber green mottle mosaic virus (CGMMV) belongs to the Tobamovirus
   genus and is a major global plant virus on cucurbit plants. It causes
   severe disease symptoms on infected watermelon plants (Citrullus
   lanatus), particularly inducing fruit decay. However, little is known
   about the molecular mechanism of CGMMV-induced watermelon fruit decay.
   For this study, comparative analysis of transcriptome profiles of
   CGMMV-inoculated and mock-inoculated watermelon fruits were conducted
   via RNA-Seq. A total of 1,621 differently expressed genes (DEGs) were
   identified in CGMMV-inoculated watermelon, among which 1,052 were
   up-regulated and 569 were down-regulated. Functional annotation
   analysis showed that several DEGs were involved in carbohydrate
   metabolism, hormone biosynthesis and signaling transduction, secondary
   metabolites biosynthesis, and plant-pathogen interactions. We
   furthermore found that some DEGs were related to cell wall components
   and photosynthesis, which may directly be involve in the development of
   the symptoms associated with diseased watermelons. To confirm the
   RNA-Seq data, 15 DEGs were selected for gene expression analysis by
   qRT-PCR. The results showed a strong correlation between these two sets
   of data. Our study identified many candidate genes for further
   functional studies during CGMMV-watermelon interactions, and will
   furthermore help to clarify the understanding of pathogenic mechanism
   underlying CGMMV infection in cucurbit plants.

Introduction

   Cucumber green mottle mosaic virus (CGMMV) is a member of the
   Tobamovirus genus in the Virgaviridae family and is an important viral
   pathogen of cucurbit crops worldwide^[32]1–[33]4. CGMMV usually
   produces typical mosaic patterning on the leaves of infected plants and
   causes fruits deformation^[34]5,[35]6, and ultimately results in yield
   reduction and considerable economic losses^[36]1,[37]7,[38]8. CGMMV was
   first reported from the UK in 1935^[39]9 and has subsequently rapidly
   spread to several countries, including Greece^[40]10, Japan^[41]11,
   Korea^[42]12, Israel^[43]1, Pakistan^[44]13, India^[45]14,
   America^[46]15 and Canada^[47]16. In China, the occurrence of CGMMV in
   watermelon-planting areas was first reported in 2006^[48]17 and has now
   been found in many provinces and regions across China. It usually
   causes severe disease symptoms on infected watermelon plants,
   especially inducing fruit decay, which is also called ‘blood flesh’,
   where the inner pulp transforms to water-soaked dirty red and even
   flesh acidulated^[49]4,[50]11,[51]18. China is the largest watermelon
   producer of the world and occupied almost 80% of the total global
   watermelon production in 2014 ([52]http://faostat.fao.org/).
   Considering the potential threat to the production of cucurbit crops,
   in May 2007 CGMMV has been listed as a quarantine pest by the Chinese
   government^[53]19. Many researches studied disease surveys, molecular
   detection, and sequence analysis of CGMMV on
   watermelons^[54]11,[55]12,[56]17,[57]20. However, the molecular
   mechanism of watermelon fruit decay caused by CGMMV still remains
   unclear.

   High-throughput sequencing techniques, such as RNA-Seq, provide a
   powerful tool to investigate the global transcriptome changes of plants
   in response to pathogen infection. Several virus-plant interaction
   analyses have been performed at the transcriptional level to study both
   the physiological and metabolic processes variations of infected
   plants^[58]21–[59]23. Virus-derived small interfering RNAs (vsiRNAs)
   profiles of CGMMV-infected cucumber^[60]24 and different tissues from
   bottle gourd^[61]25 have been investigated via high-throughput
   sequencing, and the results indicated that the characteristics of the
   vsiRNAs between different host plants or tissues were distinctly
   different. Eight novel and 23 known miRNAs have been identified that
   cucumber leaves produced in response to CGMMV infection, which is
   useful to help elucidate host-pathogen interactions as well as to
   screen for cucumber resistance genes^[62]6. Recently, the miRNAs
   sequencing analysis of watermelon leaves infected by CGMMV revealed
   that target genes for CGMMV-responsive miRNAs were involved in cell
   wall modulation, plant hormone signaling, primary and secondary
   metabolism, and intracellular transport^[63]26. Furthermore, several
   transcriptome analyses of watermelon fruits during development and
   ripening^[64]27,[65]28 provided meaningful references to further