Turning Waste into Beneficial Resource: Implication of Ageratum conyzoides L. in Sustainable Agriculture, Environment and Biopharma Sectors.

The annual herb, Ageratum conyzoides L. (Asteraceae), is distributed throughout the world. Although invasive, it can be very useful as a source of essential oils, pharmaceuticals, biopesticides, and bioenergy. However, very limited information exists on the molecular basis of its different utility as previous investigations were mainly focused on phytochemical/biological activity profiling. Here we have explored various properties of A. conyzoides that may offer environmental, ecological, agricultural, and health benefits. As this aromatic plant harbors many important secondary metabolites that may have various implications, biotechnological interventions such as genomics, metabolomics and tissue-culture can be indispensable tools for their mass-production. Further, A. conyzoides acts as a natural reservoir of begomoviruses affecting a wide range of plant species. As the mechanisms of disease spreading and crop infection are not fully clear, whole-genome sequencing and various advanced molecular technologies including RNAi, CRISPER/Cas9, multi-omics approaches, etc., may aid to decipher the molecular mechanism of such disease development and thus, can be useful in crop protection. Overall, improved knowledge of A. conyzoides is not only essential for developing sustainable weed control strategy but can also offer potential ways for biomedicinal, environment, safe and clean agriculture applications.

Complete nucleotide sequence of a novel monopartite begomovirus infecting Ageratum conyzoides in China.

Two isolates of a novel monopartite begomovirus were obtained from naturally infected Ageratum conyzoides plants showing typical leaf curling and enation symptoms in Sichuan Province, China. The complete DNA sequences of two isolates were determined to be 2749 nucleotides in length. Sequence analysis showed that the two isolates shared 99.5% identity, and the highest identity (89.5-89.6%) was with the DNA sequence of tomato leaf curl Hainan virus (ToLCHaiV). No other begomoviruses or satellite molecules were detected in the two samples. Based on the species demarcation criterion for the genus Begomovirus established by the Geminiviridae Study Group, the virus is a novel monopartite begomovirus, and the tentative name "ageratum leaf curl Sichuan virus" (ALCScV) is proposed. Phylogenetic analysis showed that it clustered with ToLCHaiV, and recombination analysis showed that ALCScV might have arisen by recombination between viruses related to ToLCHaiV, ageratum leaf curl virus (ALCuV), and sida leaf curl virus (SiLCuV).

Characterization of the cryptic AV3 promoter of ageratum yellow vein virus in prokaryotic and eukaryotic systems.

A cryptic prokaryotic promoter, designated AV3 promoter, has been previously identified in certain begomovirus genus, including ageratum yellow vein virus isolate NT (AYVV-NT). In this study, we demonstrated that the core nucleotides in the putative -10 and -35 boxes are necessary but not sufficient for promoter activity in Escherichia coli, and showed that AYVV-NT AV3 promoter could specifically interact with single-stranded DNA-binding protein and sigma 70 of E. coli involved in transcription. Several AYVV-NT-encoded proteins were found to increase the activity of AV3 promoter. The transcription start sites downstream to AV3 promoter were mapped to nucleotide positions 803 or 805 in E. coli, and 856 in Nicotiana benthamiana. The eukaryotic activity of AV3 promoter and the translatability of a short downstream open reading frame were further confirmed by using a green fluorescent protein reporter construct in yeast (Saccharomyces cerevisiae) cells. These results suggested that AV3 promoter might be a remnant of evolution that retained cryptic activity at present.

Mixed infection by two West African tomato-infecting begomoviruses and ageratum leaf curl Cameroon betasatellite in tomato in Cameroon.

Begomovirus isolates ToF3B2 and ToF3B17 and betasatellite isolate SatBToF3 were obtained from the same infected tomato plant showing begomovirus disease symptoms in Fontem, Cameroon. The full-length nucleotide sequences of ToF3B2, ToF3B17 and SatBToF3 were cloned and sequenced and were determined to be 2,797 nt, 2,794 and 1,373 nt long respectively. When compared with other begomovirus and betasatellite sequences, ToF3B2 was 93.5 % identical to Tomato leaf curl Togo virus, ToF3B17 was 95 % identical to Tomato leaf curl Cameroon virus and SatBToF3 was 92 % identical to Ageratum leaf curl Cameroon betasatellite (ALCCMB), respectively. The identification of ALCCMB in Ageratum and now in tomato strongly suggests Ageratum may be an alternative host to these viruses and that ALCCMB is non host specific and may cause severe diseases when transmitted to other crops.

Ageratum conyzoides: a host to a unique begomovirus disease complex in Cameroon.

Ageratum conyzoides (goat weed) is a widespread uncultivated species in Cameroon that exhibits leaf curl disease (LCD) symptoms suggestive of begomovirus infection. In Asia, different begomovirus-satellite complexes have been identified in A. conyzoides. The objective of this study was to determine the identity of the suspect begomoviruses and their associated satellites in A. conyzoides in Cameroon. The results indicated that all three symptomatic A. conyzoides plants examined were infected with a new begomovirus species, herein named Ageratum leaf curl Cameroon virus (ALCCMV). The ALCCMV genome sequences shared their highest identity, at 84.3-88.5%, with a group of tomato-infecting begomoviruses from West Africa. In addition, a betasatellite and an alphasatellite were cloned from the same symptomatic A. conyzoides plants. The betasatellite sequences shared limited sequence identity at 37% or less with the betasatellite Cotton leaf curl Gezira betasatellite, and the new betasatellite species is herein named Ageratum leaf curl Cameroon betasatellite (ALCCMB). The alphasatellite shared 80% nt identity with Tomato leaf curl Cameroon alphasatellite (ToLCCMA), and the new alphasatellite species is herein named Ageratum leaf curl Cameroon alphasatellite (ALCCMA). In addition, two fragments containing begomovirus-alphasatellite sequences were cloned from sample AGLI4, and they were related to the defecting interfering molecule (Y14167) associated with Ageratum yellow vein virus from Asia. These results suggest that the begomoviral-satellite complexes infecting A. conyzoides in Cameroon may be as complex or more so, to species and strains reported thus far from Asia.

Characterization of alphasatellites associated with monopartite begomovirus/betasatellite complexes in Yunnan, China.

BACKGROUND: Alphasatellites are single-stranded molecules that are associated with monopartite begomovirus/betasatellite complexes. RESULTS: Alphasatellites were identified in begomovirus-infected plant samples in Yunnan, China. All samples that contained alphasatellites also contained betasatellites, but only some samples that contained betasatellites contained alphasatellites. Thirty-three alphasatellites were sequenced, and they ranged from 1360 to 1376 nucleotides. All alphasatellites contain 3 conserved features: a single open reading frame (Rep), a conserved hairpin structure, and an adenine-rich (A-rich) region. On the basis of the phylogenetic tree of the complete nucleotide sequences, the alphasatellites were divided into 3 types with one exception. Type 1 was associated with Tomato yellow leaf curl China virus (TYLCCNV)/Tomato yellow leaf curl China betasatellite (TYLCCNB) complex. Type 2 was associated with Tobacco curly shoot virus (TbCSV)/Tobacco curly shoot betasatellite (TbCSB) complex. Type 3 was associated with TbCSV/Ageratum yellow vein betasatellite (AYVB) complex. Within each type, nucleotide sequence identity ranged from 83.4 to 99.7%, while 63.4-81.3% identity was found between types. Mixed infections of alphasatellites associated with begomovirus/betasatellite complexes were documented. CONCLUSIONS: Our results validate that alphasatellites are only associated with begomovirus/betasatellite complexes. Thirty-three sequenced alphasatellites isolated from Yunnan Province, China were divided into 3 types--each associated with a specific begomovirus/betasatellite complex. Mix-infections of alphasatellite molecules may not be unusual.

Identification of the virulence factors and suppressors of posttranscriptional gene silencing encoded by Ageratum yellow vein virus, a monopartite begomovirus.

Ageratum yellow vein disease (AYVD) is caused by the association of a Tomato leaf curl Java betasatellite [Indonesia:Indonesia 1:2003] (ToLCJB-[ID:ID1:03]) with a begomovirus component. Our previous results demonstrated that ToLCJB-[ID:ID:03] is essential for induction of leaf curl symptoms in plants and transgene expression of its betaC1 gene in Nicotiana benthamiana plants induces virus-like symptoms. Here we show that Ageratum yellow vein virus-Indonesia [Indonesia: Tomato] (AYVV-ID[ID:Tom]) alone could systemically infect the plants and induced upward leaf curl symptoms. ToLCJB-[ID:ID1:03] was required, in addition to AYVV-ID[ID:Tom], for induction of severe downward leaf curl disease in N. benthamiana plants. However, DNAbeta01fsbetaC1, which encompasses a frameshift mutation, did not induce severe symptoms in N. benthamiana when co-inoculated with AYVV-ID[ID:Tom]. The infectivity analysis of AYVV-ID[ID:Tom] and its associated betasatellite encoded genes using Potato virus X (PVX) vector were carried out in N. benthamiana, indicate that the V2 and betaC1 genes are symptom determinants. We have identified the DNA encoded V2 and its betasatellite, ToLCJB-[ID:ID1:03], encoded betaC1 proteins as efficient silencing suppressors of posttranscriptional gene silencing (PTGS) by using an Agrobacterium co-infiltration or heterologous PVX vector assays. However, the results also showed weak suppression of gene silencing activities for C2 and C4 induced by GFP and mRNA associated with GFP was detected. Furthermore, confocal imaging analysis of ToLCJB-[ID:ID1:03] betaC1 in the epidermal cells of N. benthamiana shows that this protein is accumulated towards the periphery of the cell and around the nucleus, however, V2 accumulated in the cell cytoplasm, C4 associated with plasma membrane and C2 exclusively targeted into nucleus. In this study, we identified as many as four distinct suppressors of RNA silencing encoded by AYVV-ID[ID:Tom] and its cognate betasatellite in the family Geminiviridae, counteracting innate antiviral response.

Strains of a new bipartite begomovirus, pepper yellow leaf curl Indonesia virus, in leaf-curl-diseased tomato and yellow-vein-diseased ageratum in Indonesia.

The complete nucleotide sequences of begomoviruses from pepper with leaf curl and yellowing symptoms, tomato with leaf curl symptoms, and ageratum with yellow vein in Indonesia were determined. On the basis of genome organization and sequence homology, they were proposed to belong to a new species, Pepper yellow leaf curl Indonesia virus (PepYLCIV), which includes the new strains PepYLCIV-Tomato and PepYLCIV-Ageratum. These viruses had bipartite genomes. Pepper virus DNAs from Indonesia (PepYLCIV, PepYLCIV-Tomato and PepYLCIV-Ageratum DNA-As) were noticeably distinct, forming a separate branch from the viruses infecting pepper. Considerable divergence was observed in the common region (CR) of the genomic components of PepYLCIV (77%), PepYLCIV-Tomato (82%) and PeYLCIV-Ageratum (75%). A stem-loop-forming region and a Rep-binding motif were identical in the CR of the three viruses. The CRs of PepYLCIV-Ageratum DNA-A was approximately 10 nucleotides longer than that of PepYLCIV DNA-A and PepYLCIV-Tomato DNA-A. A similar insertion was also found in the CR of PepYLCIV-Ageratum DNA-B. PepYLCIV DNA-A alone was infectious in pepper and Nicotiana benthamiana plants, and association with DNA-B increased symptom severity.

Replication promiscuity of DNA-beta satellites associated with monopartite begomoviruses; deletion mutagenesis of the Ageratum yellow vein virus DNA-beta satellite localizes sequences involved in replication.

Pseudorecombination studies in Nicotiana benthamiana demonstrate that Ageratum yellow vein virus (AYVV) and Eupatorium yellow vein virus (EpYVV) can functionally interact with DNA-beta satellites associated with AYVV, EpYVV, cotton leaf curl Multan virus (CLCuMV) and honeysuckle yellow vein virus (HYVV). In contrast, CLCuMV shows some specificity in its ability to interact with distinct satellites and HYVV is able to interact only with its own satellite. Using an N. benthamiana leaf disk assay, we have demonstrated that HYVV is unable to trans-replicate other satellites. To investigate the basis of trans-replication compatibility, deletion mutagenesis of AYVV DNA-beta has been used to localize the origin of replication to approximately 360 nt, encompassing the ubiquitous nonanucleotide/stem-loop structure, satellite conserved region (SCR) and part of the intergenic region immediately upstream of the SCR. Additional deletions within this intergenic region have identified a region that is essential for replication. The capacity for DNA-beta satellites to functionally interact with distinct geminivirus species and its implications for disease diversification are discussed.

A begomovirus associated with Ageratum yellow vein disease in Indonesia: evidence for natural recombination between tomato leaf curl Java virus and Ageratum yellow vein virus-[Java].

A begomovirus (2747 nucleotides) and a satellite DNA beta component (1360 nucleotides) have been isolated from Ageratum conyzoides L. plants with yellow vein symptoms growing in Java, Indonesia. The begomovirus is most closely related to Tomato leaf curl Java virus (ToLCJV) (91 and 98% in the total nucleotide and coat protein amino acid sequences, respectively), although the products of ORFs C1 and C4 are more closely related to those of Ageratum yellow vein virus-[Java] (91 and 95% identity, respectively). For this reason, the begomovirus it is considered to be a strain of ToLCJV and is referred to as ToLCJV-Ageratum. The virus probably derives from a recombination event in which nucleotides 2389-2692 of ToLCJV have been replaced with the corresponding region of the AYVV-[Java] genome, which includes the 5' part of the intergenic region and the C1 and C4 ORFs. Infection of A. conyzoides with ToLCJV-Ageratum alone produced no symptoms, but co-infection with DNAbeta induced yellow vein symptoms. Symptoms induced in Nicotiana benthamiana by ToLCJV-Ageratum, ToLCJV and AYVV-[Java] are consistent with the exchange of pathogenicity determinant ORF C4 during recombination.

The DNA beta satellite component associated with ageratum yellow vein disease encodes an essential pathogenicity protein (betaC1).

Ageratum yellow vein disease (AYVD) is caused by the geminivirus ageratum yellow vein virus (AYVV) and an associated DNA beta satellite. We have mapped a DNA beta transcript to a highly conserved open reading frame (betaC1 ORF). The most abundant transcript 5'-terminus is located 8 bases upstream of the betaC1 ORF putative initiation codon while the transcript terminates at multiple sites downstream from the putative termination codon. Disruption of betaC1 protein expression by the introduction of an internal nonsense codon prevented infection of the AYVV-satellite complex in ageratum and altered the phenotype in Nicotiana benthamiana to that produced by AYVV alone although the mutant was maintained in systemically infected tissues. Modification of the putative initiation codon to a nonsense codon produced an intermediate phenotype in N. benthamiana and a mild yellow vein phenotype in ageratum, suggesting that betaC1 protein expression could be initiated from an alternative site. N. benthamiana plants containing a dimeric DNA beta transgene produced severe developmental abnormalities, vein-greening, and cell proliferation in the vascular bundles. Expression of betaC1 protein from a potato virus X (PVX) vector also induced abnormal plant growth. Our results demonstrate that the satellite encodes at least one protein that plays a major role in symptom development and is essential for disease progression in ageratum, the natural host of the AYVD complex.

Subviral DNAs associated with geminivirus disease complexes.

Ageratum conyzoides (ageratum) is a common and widespread weed species that may act as a reservoir host for geminivirus diseases. Ageratum plants growing in Singapore and exhibiting yellow vein disease are infected with a complex mixture of viral, subviral and recombinant DNA components. This whitefly-transmitted disease is caused by the monopartite begomovirus ageratum yellow vein virus (AYVV) in association with a recently discovered satellite component referred to as DNA beta. Diseased plants also contain a subviral component, referred to as DNA 1, that has probably become associated with the begomovirus and adapted to whitefly transmission during mixed infection with an aphid-transmitted nanovirus. Unlike DNA beta, the nanovirus-like component is not essential for the disease. Recombination between the viral and subviral DNAs occurs frequently and may play an important evolutionary role by generating component diversity. The identification of a similar complex associated with cotton leaf curl disease (CLCuD), a serious constraint to cotton growing in Pakistan, and the isolation of DNA beta homologues from diverse plant species growing in widespread geographical locations suggests that such disease complexes are common and may have a significant impact on agriculture in the eastern hemisphere.

Universal primers for the PCR-mediated amplification of DNA 1: a satellite-like molecule associated with begomovirus-DNA beta complexes.

DNA 1 is a single-stranded DNA molecule of approximately 1370 nucleotides. It is associated with monopartite geminiviruses of the genus Begomovirus, which require a DNA beta component for symptomatic infection. The DNA 1 molecule requires the helper begomovirus for movement in plants, but is capable of self-replication. We designed two abutting primer pairs (DNA101/DNA102 and UN101/UN102) to conserved sequences of DNA 1. This allowed polymerase chain reaction-mediated amplification of the full-length molecule from total nucleic acid extracts produced from various host plants from geographically distinct, worldwide locations. These primers are useful both as diagnostic probes and for producing full-length infectious clones for in planta studies.