Genotype Sequencing and Phylogenetic Analysis Revealed the Origins of Citrus Yellow Vein Clearing Virus California Isolates.

The Citrus yellow vein clearing virus (CYVCV) causes a viral disease that has been reported in some citrus-growing regions in countries in Eurasia including Pakistan, India, Türkiye, Iran, China, and South Korea. Recently, CYVCV was detected in a localized urban area in a town in the middle of California's citrus-growing region and marks the first occurrence of the virus in North America. CYVCV has been reported to be spread by aphid and whitefly vectors and is graft and mechanically transmitted. Hence, it is an invasive pathogen that presents a significant threat to the California citrus industry, especially lemons, which are highly symptomatic to CYVCV. To elucidate the origin of the CYVCV California strain, we used long-read sequencing technology and obtained the complete genomes of three California CYVCV isolates, CA1, CA2, and CA3. The sequences of these isolates exhibited intergenomic similarities ranging from 95.4% to 97.4% to 54 publicly available CYVCV genome sequences, which indicated a relatively low level of heterogeneity. However, CYVCV CA isolates formed a distinct clade from the other isolates when aligned against other CYVCV genomes and coat protein gene sequences as shown by the neighbor network analysis. Based on the rooted Maximum Likelihood phylogenetic trees, CYVCV CA isolates shared the most recent common ancestor with isolates from India/South Asia. Bayesian evolutionary inferences resulted in a spatiotemporal reconstruction, suggesting that the CYVCV CA lineage diverged from the Indian lineage possibly around 1995. This analysis placed the origin of all CYVCV to around 1990, with South Asia and/or Middle East as the most plausible geographic source, which matches to the first discovery of CYVCV in Pakistan in 1988. Moreover, the spatiotemporal phylogenetic analysis indicated an additional virus diffusion pathway: one from South Asia to China and South Korea. Collectively, our phylogenetic inferences offer insights into the probable dynamics of global CYVCV dissemination, emphasizing the need for citrus industries and regulatory agencies to closely monitor citrus commodities crossing state and international borders.

VILLIN2 regulates cotton defense against Verticillium dahliae by modulating actin cytoskeleton remodeling.

The active structural change of actin cytoskeleton is a general host response upon pathogen attack. This study characterized the function of the cotton (Gossypium hirsutum) actin-binding protein VILLIN2 (GhVLN2) in host defense against the soilborne fungus Verticillium dahliae. Biochemical analysis demonstrated that GhVLN2 possessed actin-binding, -bundling, and -severing activities. A low concentration of GhVLN2 could shift its activity from actin bundling to actin severing in the presence of Ca2+. Knockdown of GhVLN2 expression by virus-induced gene silencing reduced the extent of actin filament bundling and interfered with the growth of cotton plants, resulting in the formation of twisted organs and brittle stems with a decreased cellulose content of the cell wall. Upon V. dahliae infection, the expression of GhVLN2 was downregulated in root cells, and silencing of GhVLN2 enhanced the disease tolerance of cotton plants. The actin bundles were less abundant in root cells of GhVLN2-silenced plants than in control plants. However, upon infection by V. dahliae, the number of actin filaments and bundles in the cells of GhVLN2-silenced plants was raised to a comparable level as those in control plants, with the dynamic remodeling of the actin cytoskeleton appearing several hours in advance. GhVLN2-silenced plants exhibited a higher incidence of actin filament cleavage in the presence of Ca2+, suggesting that pathogen-responsive downregulation of GhVLN2 could activate its actin-severing activity. These data indicate that the regulated expression and functional shift of GhVLN2 contribute to modulating the dynamic remodeling of the actin cytoskeleton in host immune responses against V. dahliae.

Blaze a New Trail: Plant Virus Xylem Exploitation.

Viruses are trailblazers in hijacking host systems for their own needs. Plant viruses have been shown to exploit alternative avenues of translocation within a host, including a challenging route through the xylem, to expand their niche and establish systemic spread, despite apparent host-imposed obstacles. Recent findings indicate that plant viruses from many families could successfully hack xylem cells in a broad range of plant hosts, including herbaceous and perennial woody plants. Similar to virus-related structures present in the phloem, virus particles and membrane-containing viral replication complexes are often observed in the xylem. Except for a few single-stranded DNA viruses in the family Geminiviridae and a negative-sense single-stranded RNA rhabdovirus, Lettuce necrotic yellows virus, the majority of the viruses that were detected in the xylem belong to the group of positive-sense RNA viruses. The diversity of the genome organization and virion morphology of those viruses indicates that xylem exploitation appears to be a widely adapted strategy for plant viruses. This review outlines the examples of the xylem-associated viruses and discusses factors that regulate virus inhabitation of the xylem as well as possible strategies of virus introduction into the xylem. In some cases, plant disease symptoms have been shown to be closely related to virus colonization of the xylem. Inhibiting viral xylem invasion could raise potential attractive approaches to manage virus diseases. Therefore, the identification of the host genes mediating virus interaction with the plant xylem tissue and understanding the underlying mechanisms call for more attention.

Citrus Tristeza Virus: From Pathogen to Panacea.

Citrus tristeza virus (CTV) is the most destructive viral pathogen of citrus. During the past century, CTV induced grave epidemics in citrus-growing areas worldwide that have resulted in a loss of more than 100 million trees. At present, the virus continues to threaten citrus production in many different countries. Research on CTV is accompanied by distinctive challenges stemming from the large size of its RNA genome, the narrow host range limited to slow-growing Citrus species and relatives, and the complexity of CTV populations. Despite these hurdles, remarkable progress has been made in understanding the CTV-host interactions and in converting the virus into a tool for crop protection and improvement. This review focuses on recent advances that have shed light on the mechanisms underlying CTV infection. Understanding these mechanisms is pivotal for the development of means to control CTV diseases and, ultimately, turn this virus into an ally.

Location matters: from changing a presumption about the Citrus tristeza virus tissue tropism to understanding the stem pitting disease.

Stem pitting is a common virus-induced disease phenotype that tremendously impacts growth of perennial woody plants. How stem pitting develops in the infected trees remains unclear. Here, we assessed the development of stem pits upon infection of citrus by Citrus tristeza virus (CTV), which has been regarded as 'phloem-limited'. By taking advantage of a highly susceptible virus host - Citrus macrophylla - and a CTV isolate lacking a viral effector - the p33 protein, the development pattern of stem pitting was revealed via time-course observations and histological analyses. The stem pits result from the virus-colonized nonlignified 'gumming' malformations which are initiated by virus invasion into multiple spatially separated tissue layers - protophloem, metaphloem, and, unexpectedly, metaxylem. Notably, invasion of CTV into the unspecialized metaxylem cells interrupted the differentiation of the xylem tracheary elements. With the radial spread of CTV into the adjacent cells towards the stem periphery, the clusters of virus-colonized immature metaxylem cells extended in size, merging, at a certain stage, with virus-bearing cells in the protophloem and metaphloem layers. Collectively, our data provide a new insight into the process of the stem pitting development and the role of the xylem tissue in the virus pathogenicity.

Citrus miraculin-like protein hijacks a viral movement-related p33 protein and induces cellular oxidative stress in defence against Citrus tristeza virus.

To defend against pathogens, plants have developed a complex immune system, which recognizes the pathogen effectors and mounts defence responses. In this study, the p33 protein of Citrus tristeza virus (CTV), a viral membrane-associated effector, was used as a molecular bait to explore virus interactions with host immunity. We discovered that Citrus macrophylla miraculin-like protein 2 (CmMLP2), a member of the soybean Kunitz-type trypsin inhibitor family, targets the viral p33 protein. The expression of CmMLP2 was up-regulated by p33 in the citrus phloem-associated cells. Knock-down of the MLP2 expression in citrus plants resulted in a higher virus accumulation, while the overexpression of CmMLP2 reduced the infectivity of CTV in the plant hosts. Further investigation revealed that, on the one hand, binding of CmMLP2 interrupts the cellular distribution of p33 whose proper function is necessary for the effective virus movement throughout the host. On the other hand, the ability of CmMLP2 to reorganize the endomembrane system, amalgamating the endoplasmic reticulum and the Golgi apparatus, induces cellular stress and accumulation of the reactive oxygen species, which inhibits the replication of CTV. Altogether, our data suggest that CmMLP2 employs a two-way strategy in defence against CTV infection.

A Long Non-Coding RNA of Citrus tristeza virus: Role in the Virus Interplay with the Host Immunity.

During infection, Citrus tristeza virus (CTV) produces a non-coding subgenomic RNA referred to as low-molecular-weight tristeza 1 (LMT1), which for a long time has been considered as a by-product of the complex CTV replication machinery. In this study, we investigated the role of LMT1 in the virus infection cycle using a CTV variant that does not produce LMT1 (CTV-LMT1d). We showed that lack of LMT1 did not halt virus ability to replicate or form proper virions. However, the mutant virus demonstrated significantly reduced invasiveness and systemic spread in Nicotiana benthamiana as well as an inability to establish infection in citrus. Introduction of CTV-LMT1d into the herbaceous host resulted in elevation of the levels of salicylic acid (SA) and SA-responsive pathogenesis-related genes beyond those upon inoculation with wild-type (WT) virus (CTV-WT). Further analysis showed that the LMT1 RNA produced by CTV-WT or via ectopic expression in the N. benthamiana leaves suppressed SA accumulation and up-regulated an alternative oxidase gene, which appeared to mitigate the accumulation of reactive oxygen species. To the best of our knowledge, this is the first report of a plant viral long non-coding RNA being involved in counter-acting host response by subverting the SA-mediated plant defense.

The p33 protein of Citrus tristeza virus affects viral pathogenicity by modulating a host immune response.

Accumulation of reactive oxygen species (ROS) is a general plant basal defense strategy against viruses. In this study, we show that infection by Citrus tristeza virus (CTV) triggered ROS burst in Nicotiana benthamiana and in the natural citrus host, the extent of which was virus-dose dependent. Using Agrobacterium-mediated expression of CTV-encoded proteins in N. benthamiana, we found that p33, a unique viral protein, contributed to the induction of ROS accumulation and programmed cell death. The role of p33 in CTV pathogenicity was assessed based on gene knockout and complementation in N. benthamiana. In the citrus-CTV pathosystem, deletion of the p33 open reading frame in a CTV variant resulted in a significant decrease in ROS production, compared to that of the wild type CTV, which correlated with invasion of the mutant virus into the immature xylem tracheid cells and abnormal differentiation of the vascular system. By contrast, the wild type CTV exhibited phloem-limited distribution with a minor effect on the vasculature. We conclude that the p33 protein is a CTV effector that negatively affects virus pathogenicity and suggest that N. benthamiana recognizes p33 to activate the host immune response to restrict CTV into the phloem tissue and minimize the disease syndrome.

Functional diversification upon leader protease domain duplication in the Citrus tristeza virus genome: Role of RNA sequences and the encoded proteins.

Viruses from the family Closteroviridae show an example of intra-genome duplications of more than one gene. In addition to the hallmark coat protein gene duplication, several members possess a tandem duplication of papain-like leader proteases. In this study, we demonstrate that domains encoding the L1 and L2 proteases in the Citrus tristeza virus genome underwent a significant functional divergence at the RNA and protein levels. We show that the L1 protease is crucial for viral accumulation and establishment of initial infection, whereas its coding region is vital for virus transport. On the other hand, the second protease is indispensable for virus infection of its natural citrus host, suggesting that L2 has evolved an important adaptive function that mediates virus interaction with the woody host.

The Thioredoxin GbNRX1 Plays a Crucial Role in Homeostasis of Apoplastic Reactive Oxygen Species in Response to Verticillium dahliae Infection in Cotton.

Examining the proteins that plants secrete into the apoplast in response to pathogen attack provides crucial information for understanding the molecular mechanisms underlying plant innate immunity. In this study, we analyzed the changes in the root apoplast secretome of the Verticillium wilt-resistant island cotton cv Hai 7124 (Gossypium barbadense) upon infection with Verticillium dahliae Two-dimensional differential gel electrophoresis and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis identified 68 significantly altered spots, corresponding to 49 different proteins. Gene ontology annotation indicated that most of these proteins function in reactive oxygen species (ROS) metabolism and defense response. Of the ROS-related proteins identified, we further characterized a thioredoxin, GbNRX1, which increased in abundance in response to V. dahliae challenge, finding that GbNRX1 functions in apoplastic ROS scavenging after the ROS burst that occurs upon recognition of V. dahliae Silencing of GbNRX1 resulted in defective dissipation of apoplastic ROS, which led to higher ROS accumulation in protoplasts. As a result, the GbNRX1-silenced plants showed reduced wilt resistance, indicating that the initial defense response in the root apoplast requires the antioxidant activity of GbNRX1. Together, our results demonstrate that apoplastic ROS generation and scavenging occur in tandem in response to pathogen attack; also, the rapid balancing of redox to maintain homeostasis after the ROS burst, which involves GbNRX1, is critical for the apoplastic immune response.

The RING finger protein NtRCP1 is involved in the floral transition in tobacco (Nicotiana tabacum).

The transition from the vegetative phase to the reproductive phase is a major developmental process in flowering plants. The underlying mechanism controlling this cellular process remains a research focus in the field of plant molecular biology. In the present work, we identified a gene encoding the C3H2C3-type RING finger protein NtRCP1 from tobacco BY-2 cells. Enzymatic analysis demonstrated that NtRCP1 is a functional E3 ubiquitin ligase. In tobacco plants, expression level of NtRCP1 was higher in the reproductive shoot apices than in the vegetative ones. NtRCP1-overexpressing plants underwent a more rapid transition from the vegetative to the reproductive phase and flowered markedly earlier than the wild-type control. Histological analysis revealed that the shoot apical meristem of NtRCP1-overexpressing plants initiated inflorescence primordia precociously compared to the wild-type plant due to accelerated cell division. Overexpression of NtRCP1 in BY-2 suspension cells promoted cell division, which was a consequence of the shortened G2 phase in the cell cycle. Together, our data suggest that NtRCP1 may act as a regulator of the phase transition, possibly through its role in cell cycle regulation, during vegetative/reproductive development in tobacco plant.