Plant architecture and leaf color are important factors influencing cotton fiber yield. In this study, based on genetic analysis, stem paraffin sectioning, and phytohormone treatments, we showed that the dwarf-red (DR) cotton mutant is a gibberellin-sensitive mutant caused by a mutation in a single dominant locus, designated GhDR. Using bulked segregant analysis (BSA) and genotyping by target sequencing (GBTS) approaches, we located the causative mutation to a ~197-kb genetic interval on chromosome A09 containing 25 annotated genes. Based on gene annotation and expression changes between the mutant and normal plants, GH_A09G2280 was considered to be the best candidate gene responsible for the dwarf and red mutant phenotypes. A 2-nucleotide deletion was found in the coding region of GhDR/GH_A09G2280 in the DR mutant, which caused a frameshift and truncation of GhDR. GhDR is a homolog of Arabidopsis AtBBX24, and encodes a B-box zinc finger protein. The frameshift deletion eliminated the C-terminal nuclear localization domain and the VP domain of GhDR, and altered its subcellular localization. A comparative transcriptome analysis demonstrated downregulation of the key genes involved in gibberellin biosynthesis and the signaling transduction network, as well as upregulation of the genes related to gibberellin degradation and the anthocyanin biosynthetic pathway in the DR mutant. The results of this study revealed the potential molecular basis by which plant architecture and anthocyanin accumulation are regulated in cotton.  

Citrus yellow mottle-associated virus (CiYMaV) belonging to the subgenus Mandarivirus within the genus Potexvirus, was first identified in 2018 from Pakistan (CiYMaV-PK), where it is endemic in several regions.  Here, three full-length cDNA clones (pCiYMaV-FL-1, pCiYMaV-FL-18, and pCiYMaV-FL-22) corresponding to the genomic RNA of CiYMaV were constructed and then agroinfiltrated on Chandler pummelo (Citrus grandis) seedlings using the vacuum infiltration method.  All the inoculated plants developed severe vein yellowing, leaf mottling, and dwarfing symptoms by 40 days post-infiltration (dpi).  The results of a direct tissue blot immunoassay and reverse transcription polymerase chain reaction detection showed 94.7–100% infection rates of pCiYMaV-FL at 60 dpi.  Despite there being no observed difference among the three clones in the severity of symptom, pCiYMaV-FL-22 showed the highest accumulation levels of viral RNA and coat proteins.  Moreover, pCiYMaV-FL-22 successfully infected seven other citrus varieties and induced symptoms in five of them.  Transmission electron microscopy identified the presence of filamentous virus particles in extracts from systemic leaves of the plants infected with pCiYMaV-FL-22 at 6-months post-infiltration.  Taken together, the results indicate that Koch’s postulates were fulfilled for CiYMaV in citrus plants.  This is the first report of full-length infectious cDNA clones of CiYMaV, and thus, the data provide a basis for further study of the molecular mechanisms of virus infection and the development of a viral vector to express foreign genes in citrus plants.

‘Candidatus Liberibacter asiaticus (CLas)’, which causes citrus Huanglongbing (HLB) disease, has not been successfully cultured in vitro to date. Here, a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected ‘Changyecheng’ sweet orange (Citrus sinensis Osbeck). Firstly, stem segments with a single axillary bud were cultured in vitro to allow CLas to multiply in the regenerating axillary buds. A high CLas titer was detected in the regenerated shoots on an optimized medium at 30 days after germination (DAG), and it was 28.2-fold higher than in the midribs from CLas-infected trees growing in the greenhouse. To minimize contamination during in vitro regeneration, CLas-infected axillary buds were micrografted onto seedlings of ‘Changyecheng’ sweet orange and cultured in a liquid medium. In this culture, the titers of CLas in regenerated shoots rapidly increased from 7.5×10⁴ to 1.4×10⁸ cells μg^-1 of citrus DNA during the first 40 DAG. The percentages of shoots with >1×10⁸ CLas cells μg^-1 DNA were 30% and 40% at 30 and 40 DAG, respectively. Direct tissue blot immune assay (DTBIA) indicated that the distribution of CLas was much more uniform in regenerated plantlets than in CLas-infected trees growing in the greenhouse. The disease symptoms in the plantlets were die-back, stunted growth, leaf necrosis/yellowing, and defoliation. The death rate of the plantlets was 82.0% at 60 DAG. Our results show that CLas can effectively multiply in in vitro culture. This method will be useful for studying plant–HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus.

Citrus Huanglongbing (HLB, yellow shoot disease) is one of the most serious citrus diseases worldwide.  To better improve the detection sensitivity, a droplet digital PCR (ddPCR) assay was developed for the rapid detection of ‘Candidatus Liberibacter asiaticus’ (Las), the putative causal agent of HLB.  The detection of sensitivity comparison using positive plasmid indicated that ddPCR was superior to quantitative PCR (qPCR) for detecting and quantifying Las at low concentrations.  The Las detection of 40 field samples also showed that six of 13 asymptomatic samples (46.15%) with high C_t value (>35) were positive by ddPCR.  This methodology showed great potential for early HLB infection diagnosis.

To identity the potential pathogen associated with the yellow vein clearing symptom on lemon trees, the profiles of virus-derived small interfering RNAs from citrus samples were obtained and analyzed by deep sequencing method in this study.  Twenty-seven contigs almost cover the full length genome of Citrus yellow vein clearing virus (CYVCV) isolate YN were obtained using the small RNA deep sequencing technology.  Analysis showed that this isolate CQ shared the highest nucleotide identity with isolate Y1 (JX040635) and YN (KP313242), both of which belong to the genus Mandarivirus in the family Alphaflexiviridae.  Mapping analysis of viral-derived siRNA (vsiRNA) profiles revealed an uneven distribution pattern of their generations along both positive and negative genome strands, and 22- and 21-nt vsiRNAs ranked the majority.  BLAST against viroids and other viral databases confirmed that this sample was single-infected only by CYVCV, which indicated that CYVCV was the exact causal agent for the yellow clearing symptom occurring on lemon.  This is the first CYVCV isolate detected in Chongqing and the second in China.  This result could provide a molecular basis for the investigation of citrus viral diseases to protect citrus health in this region. 

Two miniature inverted-repeat transposable elements (MITEs), MCLas-A and MCLas-B, were recently identified from ‘Candidatus Liberibacter asiaticus’ known to be associated with citrus Huanglongbing (HLB, yellow shoot disease). MCLas-A was suggested as an active MITE because of its mobility. The immediate upstream gene of the two MITEs was predicted to be a putative transposase. The goal of this study is to analyze the sequence variation in the upstream putative transposase of MITEs and explore the possible correlation between sequence variation of transposase gene and MITE activity. PCR and sequence analysis showed that 12 sequence types were found in six major amplicon types from 43 representative ‘Ca. L. asiaticus’ isolates from China, the United States and Brazil. Out of the 12 sequence types, three (T4, T5-2, T6) were reported for the first time. Recombination events were found in the two unique sequence types (T5-2 and T6) which were detected in all Brazilian isolates. Notably, no sequence variation or recombination events were detected in the upstream putative transposase gene of MCLas-A, suggesting the conservation of the transposase gene might be closely related with the MITE activity. Phylogenetic analysis demonstrated two well supported clades including five subclades were identified, clearly reflecting the geographical origins of isolates, especially that of Ruili isolates, São Paulo isolates and a few Florida isolates.