The C4 photosynthesis bifunctional enzymes, PDRPs, of maize are co-opted to cytoplasmic viral replication complexes to promote infection of a prevalent potyvirus sugarcane mosaic virus.

In maize, two pyruvate orthophosphate dikinase (PPDK) regulatory proteins, ZmPDRP1 and ZmPDRP2, are respectively specific to the chloroplast of mesophyll cells (MCs) and bundle sheath cells (BSCs). Functionally, ZmPDRP1/2 catalyse both phosphorylation/inactivation and dephosphorylation/activation of ZmPPDK, which is implicated as a major rate-limiting enzyme in C4 photosynthesis of maize. Our study here showed that maize plants lacking ZmPDRP1 or silencing of ZmPDRP1/2 confer resistance to a prevalent potyvirus sugarcane mosaic virus (SCMV). We verified that the C-terminal domain (CTD) of ZmPDRP1 plays a key role in promoting viral infection while independent of enzyme activity. Intriguingly, ZmPDRP1 and ZmPDRP2 re-localize to cytoplasmic viral replication complexes (VRCs) following SCMV infection. We identified that SCMV-encoded cytoplasmic inclusions protein CI targets directly ZmPDRP1 or ZmPDRP2 or their CTDs, leading to their re-localization to cytoplasmic VRCs. Moreover, we found that CI could be degraded by the 26S proteasome system, while ZmPDRP1 and ZmPDRP2 could up-regulate the accumulation level of CI through their CTDs by a yet unknown mechanism. Most importantly, with genetic, cell biological and biochemical approaches, we provide evidence that BSCs-specific ZmPDRP2 could accumulate in MCs of Zmpdrp1 knockout (KO) lines, revealing a unique regulatory mechanism crossing different cell types to maintain balanced ZmPPDK phosphorylation, thereby to keep maize normal growth. Together, our findings uncover the genetic link of the two cell-specific maize PDRPs, both of which are co-opted to VRCs to promote viral protein accumulation for robust virus infection.

Activated malate circulation contributes to the manifestation of light-dependent mosaic symptoms.

Mosaic symptoms are commonly observed in virus-infected plants. However, the underlying mechanism by which viruses cause mosaic symptoms as well as the key regulator(s) involved in this process remain unclear. Here, we investigate maize dwarf mosaic disease caused by sugarcane mosaic virus (SCMV). We find that the manifestation of mosaic symptoms in SCMV-infected maize plants requires light illumination and is correlated with mitochondrial reactive oxidative species (mROS) accumulation. The transcriptomic and metabolomic analyses results together with the genetic and cytopathological evidence indicate that malate and malate circulation pathways play essential roles in promoting mosaic symptom development. Specifically, at the pre-symptomatic infection stage or infection front, SCMV infection elevates the enzymatic activity of pyruvate orthophosphate dikinase by decreasing the phosphorylation of threonine527 under light, resulting in malate overproduction and subsequent mROS accumulation. Our findings indicate that activated malate circulation contributes to the manifestation of light-dependent mosaic symptoms via mROS.

Sugarcane mosaic virus remodels multiple intracellular organelles to form genomic RNA replication sites.

Positive-stranded RNA viruses usually remodel the host endomembrane system to form virus-induced intracellular vesicles for replication during infections. The genus Potyvirus of the family Potyviridae represents the largest number of positive single-stranded RNA viruses, and its members cause great damage to crop production worldwide. Although potyviruses have a wide host range, each potyvirus infects a relatively limited number of host species. Phylogenesis and host range analysis can divide potyviruses into monocot-infecting and dicot-infecting groups, suggesting that they differ in their infection mechanisms, probably during replication. Comprehensive studies on the model dicot-infecting turnip mosaic virus have shown that the 6K2-induced replication vesicles are derived from the endoplasmic reticulum (ER) and subsequently target chloroplasts for viral genome replication. However, the replication site of monocot-infecting potyviruses is unknown. In this study, we show that the precursor 6K2-VPg-Pro polyproteins of dicot-infecting potyviruses and monocot-infecting potyviruses cluster phylogenetically in two separate groups. With a typical gramineae-infecting potyvirus-sugarcane mosaic virus (SCMV)-we found that replicative double-stranded RNA (dsRNA) forms aggregates in the cytoplasm but does not associate with chloroplasts. SCMV 6K2-VPg-Pro-induced vesicles colocalize with replicative dsRNA. Moreover, SCMV 6K2-VPg-Pro-induced structures target multiple intracellular organelles, including the ER, Golgi apparatus, mitochondria, and peroxisomes, and have no evident association with chloroplasts.

Maize phenylalanine ammonia-lyases contribute to resistance to Sugarcane mosaic virus infection, most likely through positive regulation of salicylic acid accumulation.

Sugarcane mosaic virus (SCMV) is a pathogen of worldwide importance that causes dwarf mosaic disease on maize (Zea mays). Until now, few maize genes/proteins have been shown to be involved in resistance to SCMV. In this study, we characterized the role of maize phenylalanine ammonia-lyases (ZmPALs) in accumulation of the defence signal salicylic acid (SA) and in resistance to virus infection. SCMV infection significantly increased SA accumulation and expression of SA-responsive pathogenesis-related protein genes (PRs). Interestingly, exogenous SA treatment decreased SCMV accumulation and enhanced resistance. Both reverse transcription-coupled quantitative PCR and RNA-Seq data confirmed that expression levels of at least four ZmPAL genes were significantly up-regulated upon SCMV infection. Knockdown of ZmPAL expression led to enhanced SCMV infection symptom severity and virus multiplication, and simultaneously resulted in decreased SA accumulation and PR gene expression. Intriguingly, application of exogenous SA to SCMV-infected ZmPAL-silenced maize plants decreased SCMV accumulation, showing that ZmPALs are required for SA-mediated resistance to SCMV infection. In addition, lignin measurements and metabolomic analysis showed that ZmPALs are also involved in SCMV-induced lignin accumulation and synthesis of other secondary metabolites via the phenylpropanoid pathway. In summary, our results indicate that ZmPALs are required for SA accumulation in maize and are involved in resistance to virus infection by limiting virus accumulation and moderating symptom severity.

One-step reverse transcription loop-mediated isothermal amplification assay for detection of Apple chlorotic leaf spot virus.

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of Apple chlorotic leaf spot virus (ACLSV). In this method, a set of four primers was designed based on the conserved regions in the coat protein gene of ACLSV, and the primers were synthesized for the RT-LAMP assay using total RNA extracted from ACLSV-infected leaf tissues. The optimal reaction temperature and assay time were determined to be 64°C and 75min, respectively. The sensitivity of RT-LAMP reactions was reliable up to a maximum dilution of 1:3125, which was more sensitive than the RT-PCR assay. The successful application of RT-LAMP to field-collected apple samples demonstrated its potential for broader applications in effectively diagnosing diseases and, consequently, its potential to control ACLSV from spreading further, particularly in many developing countries around the world. To our knowledge, this is the first application of RT-LAMP for the detection of ACLSV.

Identification of differentially regulated maize proteins conditioning Sugarcane mosaic virus systemic infection.

Sugarcane mosaic virus (SCMV) is the most important cause of maize dwarf mosaic disease. To identify maize genes responsive to SCMV infection and that may be involved in pathogenesis, a comparative proteomic analysis was performed using the first and second systemically infected leaves (termed 1 SL and 2 SL, respectively). Seventy-one differentially expressed proteins were identified in 1 SL and 2 SL upon SCMV infection. Among them, eight proteins showed the same changing patterns in both 1 SL and 2 SL. Functional annotations of regulated proteins and measurement of photosynthetic activity revealed that photosynthesis was more inhibited and defensive gene expression more pronounced in 1 SL than in 2 SL. Knockdown of regulated proteins in both 1 SL and 2 SL by a brome mosaic virus-based gene silencing vector in maize indicated that protein disulfide isomerase-like and phosphoglycerate kinase were required for optimal SCMV replication. By contrast, knockdown of polyamine oxidase (ZmPAO) significantly increased SCMV accumulation, implying that ZmPAO activity might contribute to resistance or tolerance. The results suggest that combining comparative proteomic analyses of different tissues and virus-induced gene silencing is an efficient way to identify host proteins supporting virus replication or enhancing resistance to virus infection.

A multiple RT-PCR assay for simultaneous detection and differentiation of latent viruses and apscarviroids in apple trees.

Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), and Apple stem pitting virus (ASPV) are three latent viruses frequently occurring in apple trees worldwide. In field orchards, these viruses are frequently found in a mixed infection with viroids in the genus Apscarviroid, including Apple scar skin viroid, and Apple dimple fruit viroid. Together these viruses and viroids could cause serious damage to apple fruit production worldwide. Rapid and efficient detection methods are pivotal to identify and select the virus-free propagation material for healthy apple orchard management. In this study a multiplex Reverse Transcription-PCR (RT-PCR) was developed and optimized for simultaneous detection and differentiation of the three latent viruses and apscarviroids. With newly designed specific primers for ACLSV, ASGV, APSV, and EF-1α (as an internal control), and a pair of degenerate primers for apscarviroids, optimized parameters for multiplex RT-PCR were determined. The resulting PCR products from each target virus and viroid could be easily identified because their product sizes differ by at least a 100bp. The multiplex RT-PCR method is expected to detect different variants of the viruses as the test results showed that a variety of isolates from different regions in China gave positive results. To the best of our knowledge, this multiplex RT-PCR assay is the first to simultaneously detect multiple viruses and viroids infecting apple trees in a single reaction tube. This assay, therefore, offers a useful tool for routine certification and quarantine programs.