Xiao-Feng Zhang

Plants frequently encounter multiple pathogens concurrently, yet the molecular bases of pathogen–pathogen interactions during mixed infections remain largely unresolved. Here we show that co‐infection of rice by Rice orange leaf phytoplasma (ROLP) and Rice stripe mosaic virus (RSMV) markedly suppresses RSMV proliferation in both host plants and the insect vector. Electron microscopy and confocal imaging reveal that ROLP and RSMV co‐inhabit individual rice phloem cells and undergo close membrane interaction, producing swollen RSMV virions with reduced infectivity. We further demonstrate that the ROLP membrane protein Imp interacts with the RSMV G envelope protein and competitively disrupts the G–M association essential for virion assembly. The Imp region encompassing residues 96–122 (Imp‐α4) is necessary for this inhibition. Transgenic rice overexpressing Imp or Imp‐α4 phenocopies these effects, displaying deformed virions and diminished viral load. These findings uncover a cross‐kingdom antagonistic mechanism between two phylogenetically distant pathogens and offer conceptual directions for engineering durable antiviral resistance.

RipP1 is a well-characterized avirulence effector that induces a hypersensitive response (HR) in three tobacco species. However, the molecular mechanisms by which host proteins recognize RipP1 to activate a defense response and modulate host–pathogen interactions remain largely unknown. In this study, we screened a Nicotiana benthamiana cDNA library via yeast two-hybrid assay and identified FRIGIDA-like protein 4a (FRL4a) as a host protein interacting with RipP1. Secondary structure analysis of FRL4a and construction of serial mutants revealed that the ClyA-like domain of FRL4a is the key region mediating its interaction with RipP1. Using virus-induced gene silencing (VIGS) and quantitative real-time PCR (qPCR) analysis, we found that the ability of RipP1 to induce HR was significantly attenuated in FRL4a-silenced plants, and RipP1 no longer suppressed the ethylene signaling pathway. Pathogenicity tests by inoculating R. solanacearum on N. benthamiana with different FRL4a expression levels showed enhanced bacterial wilt resistance in FRL4a-silenced plants but increased susceptibility in FRL4a-overexpressing plants. Collectively, these findings demonstrate that RipP1 suppresses the ethylene pathway through its interaction with FRL4a, and FRL4a acts as a negative regulator of tobacco resistance to bacterial wilt.

In the field, 80% of plant viruses are transmitted by insect vectors. When ingested by a sap-sucking insect such as Recilia dorsalis, persistently transmitted viruses such as rice stripe mosaic virus (RSMV) infect the gut epithelium and eventually pass to the salivary glands where they will be transmitted to the next rice (Oryza sativa) plant. To efficiently exploit insect vectors for transmission, plant viruses must overcome various immune mechanisms within the vectors, including autophagy. However, understanding how plant viruses overcome insect autophagic defenses remains limited. In this study, we provide evidence that infection with RSMV triggers an autophagic antiviral response in leafhopper cells. In this response, the G protein of RSMV binds to a leafhopper AMP-activated protein kinase (AMPK), leading to enhanced phosphorylation of Beclin-1 (BECN1), thereby inducing autophagy. Knockdown of AMPK and genes encoding members of the phosphoinositide 3-kinase (PI3K) complex composed of the autophagy-related protein 14 (ATG14), BECN1, and vacuolar protein sorting 34 (VPS34) facilitated viral infection in leafhoppers. To suppress leafhopper-induced autophagy, RSMV M protein specifically interacts with ATG14, resulting in the disintegration of PI3K complexes. This leads to reduced phosphatidylinositol-3-phosphate content and thus inhibits the G-protein- induced autophagy. Our study sheds light on the mechanism by which this rice virus evades insect autophagy antiviral defenses.

Insect melanization triggered by the conversion of prophenoloxidase to active phenoloxidase via serine proteases (SPs) is an important immediate immune response. However, how phytoplasmas evade this immune response to promote their propagation in insect vectors remains unknown. Here, we demonstrate that infection of leafhopper vectors with rice orange leaf phytoplasma (ROLP) activates the mild melanization response in hemolymph. ROLP-encoded effector protein SRP1 is highly expressed in leafhopper hemolymph, where it competitively binds to SP2, thereby inhibiting SP2-mediated cleavage of prophenoloxidase into active phenoloxidase. Consequently, microinjection of SRP1 effectively suppresses the melanization response and enhances ROLP propagation. The histidine residue at position 23 of SRP1 is essential for SRP1-SP2 interaction, and the mutation of this position abolishes its ability to inhibit such SP2-meidated cleavage, ultimately promoting melanization response and inhibiting ROLP propagation. Our findings provide insights into how phytoplasmas antagonize insect melanization response to facilitate their persistent transmission.

Rice yellow stunt virus (RYSV) P3 protein functions as a movement protein during viral infection of a plant host; its function in insect hosts remains unclear. In this study, we investigated the subcellular localization of P3 using leafhopper (Nephotettix cincticeps) cell cultures. Our results showed that P3 translocated from the cytoplasm to the nucleus in RYSV-infected leafhopper cells, where it interacted with the viral N protein as a constituent of viroplasms. Interfering with the P3 gene expression significantly suppressed viral infection in N. cincticeps. Finally, we demonstrate that the nuclear translocation of P3 in leafhopper cells depended on its interaction with RYSV N protein, which enters the nucleus via an interaction with importin α3. These findings unveil a previously unknown role for P3 in RYSV infection of the insect vector and provide valuable insights into the infection dynamics of plant rhabdoviruses.

The environmental bacterium Pseudomonas mosselii produces antagonistic secondary metabolites with inhibitory effects on multiple plant pathogens, including Ralstonia solanacearum, the causal agent of bacterial wilt. In this study, an engineered P. mosselii strain was generated to express R. solanacearum ripAA, which determines the incompatible interactions with tobacco plants. The ripAA gene, together with its native promoter, was integrated into the P. mosselii chromosome. The resulting strain showed no difference in antimicrobial activity against R. solanacearum. Promoter-LacZ fusion and RT-PCR experiments demonstrated that the ripAA gene was transcribed in culture media. Compared with that of the wild type, the engineered strain reduced the disease index by 9.1% for bacterial wilt on tobacco plants. A transcriptome analysis was performed to identify differentially expressed genes in tobacco plants, and the results revealed that ethylene- and jasmonate-dependent defense signaling pathways were induced. These data demonstrates that the engineered P. mosselii expressing ripAA can improve biological control against tobacco bacterial wilt by the activation of host defense responses.

Balakata baccata belongs to the family Euphorbiaceae and is distributed in Yunnan province, China, and other southeast Asian countries, e.g., Bangladesh, Cambodia, India, Indonesia, etc. Here, we report and characterize the complete plastome of B. baccata. The complete plastome is 163,988 bp in length and contains a typical quadripartite structure and gene content found in angiosperms, including two inverted repeat (IR) regions of 27,274 bp, a large single-copy (LSC) region of 90,946 bp and a small single-copy (SSC) region of 18,494 bp. The plastome contains 129 genes, consisting of 84 protein-coding genes, 37 tRNA genes, and eight rRNA genes. The overall G/C content in the plastome of B. baccata is 35.6%. Phylogenetic results show that B. baccata is the earliest diverging lineage of Euphorbioideae. Euphorbia helioscopia + E. esula, E. tirucalli + E. milii and B. baccata have a closer phylogenetic relationship than other taxa within Euphorbiaceae. The complete plastome sequence of B. baccata will provide a useful resource for the conservation genetics of this species as well as for phylogenetic studies in Euphorbiaceae.

IThe plastome of Manilkara zapota is found to be 158,386 bp long with the typical quadripartite structure of angiosperms, contains two inverted repeats (IRs) of 26,099 bp each, a large single-copy (LSC) region of 87,745 bp, and a small single-copy (SSC) region of 18,443 bp. The plastome contains 114 genes, consisting of 80 unique protein-coding genes, 30 unique tRNA genes, and 4 unique rRNA genes. The overall A/T content in the plastome of M. zapota is 63.00%. The phylogenetic analysis indicated that M. zapota is close to Sideroxylon wightianum within Sapotaceae in this study.

Persistently transmitted plant viruses encounter multiple membrane and tissue barriers in the process of completing their infection routes within their insect vectors. Some of these viruses have been reported to overcome the elaborate barriers of the central nervous system (CNS) to travel through the nervous tissues, but the specific mechanisms of this process remain unknown. Here, we report the axonal transport mechanism of rice yellow stunt virus (RYSV), a nucleorhabdovirus, in the CNS of the green rice leafhopper (Nephotettix cincticeps). The infection route of RYSV in the internal organs of its insect vector after ingestion of the virus was investigated by immunofluorescence microscopy. RYSV was first detected in the epithelial cells of midgut regions, from where it proceeded to the nervous system, and finally into the salivary glands. We then utilized immunofluorescence and electron microscopy to investigate the distribution of RYSV particles within the leafhopper CNS, demonstrating that non-enveloped viral particles distributed along the microtubule-based neurofilaments in the axon cytoplasm following the direct interaction of leafhopper α-tubulin with the RYSV M protein. Tubulin inhibitors inhibited the dissemination of RYSV to the CNS, then into the salivary glands in leafhoppers. We therefore describe a mechanism of plant virus transport through CNS axons as an alternative means of rapid viral dissemination in an insect vector.

In China, the rice pathogen Rice yellow stunt virus (RYSV), a member of the genus Nucleorhabdovirus in the family Rhabdoviridae, was a severe threat to rice production during the1960s and1970s. Fundamental aspects of the biology of this virus such as protein localization and formation of the RYSV viroplasm during infection of insect vector cells are largely unexplored. The specific role(s) of the structural proteins nucleoprotein (N) and phosphoprotein (P) in the assembly of the viroplasm during RYSV infection in insect vector is also unclear. In present study, we used continuous leafhopper cell culture, immunocytochemical techniques, and transmission electron microscopy to investigate the subcellular distributions of N and P during RYSV infection. Both GST pull-down assay and yeast two-hybrid assay were used to assess the in vitro interaction of N and P. The dsRNA interference assay was performed to study the functional roles of N and P in the assembly of RYSV viroplasm. Here we demonstrated that N and P colocalized in the nucleus of RYSV-infected Nephotettix cincticeps cell and formed viroplasm-like structures (VpLSs). The transiently expressed N and P are sufficient to form VpLSs in the Sf9 cells. In addition, the interactions of N/P, N/N and P/P were confirmed in vitro. More interestingly, the accumulation of RYSV was significantly reduced when the transcription of N gene or P gene was knocked down by dsRNA treatment. In summary, our results suggest that N and P are the main viral factors responsible for the formation of viroplasm in RYSV-infected insect cells. Early during RYSV infection in the insect vector, N and P interacted with each other in the nucleus to form viroplasm-like structures, which are essential for the infection of RYSV.