Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation, to promote viral infection and symptom development in N. benthamiana.

The phytohormone gibberellin (GA) is a vital plant signaling molecule that regulates plant growth and defense against abiotic and biotic stresses. To date, the molecular mechanism of the plant responses to viral infection mediated by GA is still undetermined. DELLA is a repressor of GA signaling and is recognized by the F-box protein, a component of the SCFSLY1/GID2 complex. The recognized DELLA is degraded by the ubiquitin-26S proteasome, leading to the activation of GA signaling. Here, we report that ageratum leaf curl Sichuan virus (ALCScV)-infected N. benthamiana plants showed dwarfing symptoms and abnormal flower development. The infection by ALCScV significantly altered the expression of GA pathway-related genes and decreased the content of endogenous GA in N. benthamiana. Furthermore, ALCScV-encoded C4 protein interacts with the DELLA protein NbGAI and interferes with the interaction between NbGAI and NbGID2 to prevent the degradation of NbGAI, leading to inhibition of the GA signaling pathway. Silencing of NbGAI or exogenous GA3 treatment significantly reduces viral accumulation and disease symptoms in N. benthamiana plants. The same results were obtained from experiments with the C4 protein encoded by tobacco curly shoot virus (TbCSV). Therefore, we propose a novel mechanism by which geminivirus C4 proteins control viral infection and disease symptom development by interfering with the GA signaling pathway.

First Report of Cotton Leafroll Dwarf Virus infection of Malvaviscus arboreus in China.

Cotton leafroll dwarf virus (CLRDV, genus Polerovirus, family Solemoviridae) has been reported to infect cotton in Brazil, Argentina, India, Thailand and Timor-Leste (Agrofoglio YC et al. 2017; Corrêa RL et al. 2005; Mukherjee et al. 2012; Ray et al. 2016; Sharman et al. 2015), and in the United States (Ali and Mokhtari et al. 2020; Avelar et al. 2019). It has also been recently reported to infect Cicer arietinum (chickpea) in Uzbekistan and Hibiscus syriacus in Korea (Igori et al. 2022; Kumari et al. 2020). In China, the natural infection of plants by CLRDV has not been reported previously. In August 2017, leaf samples were collected from a wild plant of Malvaviscus arboreus (Malvaceae) exhibiting symptoms including leaf yellowing and distorting in Tengchong County of Yunnan Province. Leaves were used for total RNA extraction using TRIzol Reagent (Invitrogen, USA). Small RNA library construction and deep sequencing was performed on illumina HiSeqTM 2000 platform by Novogene Bioinformatic Technology Co., Ltd (Beijing, China). A total of 11, 525, 708 raw reads were obtained and further computationally analyzed by Perl scripts. The adaptors were removed and the obtained 7, 520, 902 clean reads with size of 18- to 26-nucleotide (nt) were aligned with the GenBank virus RefSeq database using Bowtie software. These reads were mainly mapped to the genomes of hibiscus bacilliform virus (genus Badnavirus, family Caulimoviridae), hibiscus chlorotic ringspot virus (genus Betacarmovirus, family Procedovirinae), hibiscus latent Singapore virus (genus Tobamovirus, family Virgaviridae) and CLRDV isolate ARG (accession no. GU167940). The average coverage depth of clean reads mapped to CLRDV genome was 97.76%. Contigs greater than 50 nt were used to search for similar sequences by BLASTx, and 107 contigs were annotated as homologous to CLRDV isolates. To confirm CLRDV infection, reverse transcription polymerase chain reaction (RT-PCR) was performed using the specific primer pair CLRDV-F (5'-TCCACAGGAAGTATCACGTTCG-3') and CLRDV-R (5'-CCTTGTGTGGTTTGATTCGTGA-3') designed based on two of these contigs well-aligned to the genome of CLRDV isolate ARG. An amplicon of 1095-bp size was amplified, and was sequenced by Sanger sequencing (TsingKe Biological Technology, Chengdu, China), and BLASTn search results showed a maximum nucleotide identity of 95.45% with CLRDV isolate CN-S5, an isolate obtained from soybean aphid host in China (accession no. KX588248). To obtain more information on this CLRDV isolate, four primer pairs were designed and used for RT-PCR amplification (Table S1). The amplicons with size of about 860-, 1400-, 3200- and 1100-bp, were obtained separately and assembled into a complete genome sequence up to 5, 865-nt in length (isolate YN, deposited under GenBank accession no. MN057665). BLASTn showed the highest nucleotide similarity of 94.61% with CLRDV isolate CN-S5. From 2018 to 2022, additional M. arboreus samples with leaf yellowing or curling symptoms (9 from Shapingba District in Chongqing City, 5 from Nanchong City in Sichuan Province, 9 from Kunming City and 12 from Tengchong County in Yunnan Province) were collected and tested for CLRDV by RT-PCR using primer pairs CLRDV-F/CLRDV-R. The nucleotide sequences of the CLRDV P0 gene in two samples from Tengchong County were obtained by Sanger sequencing and deposited under GenBank (CLRDV isolate TCSL1 P0 gene, accession no. OQ749809; CLRDV isolate TCSW2 P0 gene, accession no. OQ749809). To our knowledge, this is the first report of CLRDV naturally infecting Malvaviscus arboreus in China, thus extending the information on its geographical distribution and host range. Malvaviscus arboreus is a widely cultivated ornamental plant in Yunnan Province, China. The natural occurrence of CLRDV not only affects the ornamental value of Malvaviscus arboreus, but also poses a potential threat to cotton production in China. This study will assist further surveillance of CLRDV infection and future development of effective protection strategies against CLRDV in China.

Tobacco curly shoot virus Down-Regulated the Expression of nbe-miR167b-3p to Facilitate Its Infection in Nicotiana benthamiana.

Tobacco curly shoot virus (TbCSV) belongs to the genus Begomovirus of the family Geminiviridae, and causes leaf curling and curly shoot symptoms in tobacco and tomato crops. MicroRNAs (miRNAs) are pivotal modulators of plant development and host-virus interactions. However, the relationship between TbCSV infection and miRNAs accumulation has not been well investigated. The present study was conducted to analyze different expressions of miRNAs in Nicotiana benthamiana in response to the infection of TbCSV via small RNAs sequencing. The results showed that 15 up-regulated miRNAs and 12 down-regulated miRNAs were differentially expressed in TbCSV infected N. benthamiana, and nbe-miR167b-3p was down-regulated. To decipher the relationship between nbe-miR167b-3p expression and the accumulations of TbCSV DNA, pCVA mediation of miRNA overexpression and PVX based short tandem target mimic (STTM) were used in this study. It was found that overexpression of nbe-miR167b-3p attenuated leaf curling symptom of TbCSV and decreased viral DNA accumulation, but suppression of nbe-miR167b-3p expression enhanced the symptoms and accumulation of TbCSV. PRCP, the target gene of nbe-miR167b-3p, was silenced in plants using VIGS and this weakened the viral symptoms and DNA accumulation of TbCSV in the plants. Overall, this study clarified the effect of nbe-miR167b-3p on plant defense during TbCSV infection, and provided a framework to reveal the molecular mechanisms of miRNAs between plants and viruses.