Draft Genome Sequence Resource for the Orange Rust Pathogen of Sugarcane Puccinia kuehnii.

Puccinia kuehnii is an obligate biotrophic fungal pathogen that causes orange rust of sugarcane, which is prevalent in many countries around the globe. In the United States, orange rust was first detected in sugarcane in Florida in 2007 and poses a persistent and economically damaging threat to the sugarcane industry in this region. Here, we generated the first genome assemblies for two isolates of P. kuehnii (1040 and 2143) collected in Florida in 2017 from two sugarcane cultivars, CL85-1040 and CP89-2143, respectively. These two rust genome resources will be of immense value for future genomic studies, particularly further exploration of the predicted secretomes that may help define key pathogenicity determinants for this economically important pathogen.

Molecular Detection and Quantification of Xanthomonas albilineans in Juice from Symptomless Sugarcane Stalks Using a Real-Time Quantitative PCR Assay.

Leaf scald, a bacterial disease caused by Xanthomonas albilineans (Ashby) Dowson, is a major limiting factor for sugarcane production worldwide. Accurate identification and quantification of X. albilineans is a prerequisite for successful management of this disease. A sensitive and robust quantitative PCR (qPCR) assay was developed in this study for detection and quantification of X. albilineans using TaqMan probe and primers targeting a putative adenosine triphosphate-binding cassette (ABC) transporter gene (abc). The novel qPCR assay was highly specific to the 43 tested X. albilineans strains belonging to different pulsed-field gel electrophoresis groups. The detection thresholds were 100 copies/µl of plasmid DNA, 100 fg/µl of bacterial genomic DNA, and 100 CFU/ml of bacterial suspension prepared from pure culture. This qPCR assay was 100 times more sensitive than a conventional PCR assay. The pathogen was detected by qPCR in 75.1% (410/546) of symptomless stalk samples, whereas only 28.4% (155/546) of samples tested positive by conventional PCR. Based on qPCR data, population densities of X. albilineans in symptomless stalks of the same varieties differed between two sugarcane production areas in China, Beihai (Guangxi Province) and Zhanjiang (Guangdong Province), and no significant correlation between these populations was identified. Furthermore, no relationship was found between these populations of the pathogen in asymptomatic stalks and the resistance level of the sugarcane varieties to leaf scald. The newly developed qPCR assay proved to be highly sensitive and reliable for the detection and quantification of X. albilineans in sugarcane stalks.

Comparative Transcriptome Profiling of Resistant and Susceptible Sugarcane Cultivars in Response to Infection by Xanthomonas albilineans.

Sugarcane (Saccharum spp. hybrids) is a major source of sugar and renewable bioenergy crop worldwide and suffers serious yield losses due to many pathogen infections. Leaf scald caused by Xanthomonas albilineans is a major bacterial disease of sugarcane in most sugarcane-planting countries. The molecular mechanisms of resistance to leaf scald in this plant are, however, still unclear. We performed a comparative transcriptome analysis between resistant (LCP 85-384) and susceptible (ROC20) sugarcane cultivars infected by X. albilineans using the RNA-seq platform. 24 cDNA libraries were generated with RNA isolated at four time points (0, 24, 48, and 72 h post inoculation) from the two cultivars with three biological replicates. A total of 105,783 differentially expressed genes (DEGs) were identified in both cultivars and the most upregulated and downregulated DEGs were annotated for the processes of the metabolic and single-organism categories, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the 7612 DEGs showed that plant-pathogen interaction, spliceosome, glutathione metabolism, protein processing in endoplasmic reticulum, and plant hormone signal transduction contributed to sugarcane's response to X. albilineans infection. Subsequently, relative expression levels of ten DEGs determined by quantitative reverse transcription-PCR (qRT-PCR), in addition to RNA-Seq data, indicated that different plant hormone (auxin and ethylene) signal transduction pathways play essential roles in sugarcane infected by X. albilineans. In conclusion, our results provide, for the first time, valuable information regarding the transcriptome changes in sugarcane in response to infection by X. albilineans, which contribute to the understanding of the molecular mechanisms underlying the interactions between sugarcane and this pathogen and provide important clues for further characterization of leaf scald resistance in sugarcane.

Occurrence of a novel mastrevirus in sugarcane germplasm collections in Florida, Guadeloupe and Réunion.

BACKGROUND: In Africa and Asia, sugarcane is the host of at least seven different virus species in the genus Mastrevirus of the family Geminiviridae. However, with the exception of Sugarcane white streak virus in Barbados, no other sugarcane-infecting mastrevirus has been reported in the New World. Conservation and exchange of sugarcane germplasm using stalk cuttings facilitates the spread of sugarcane-infecting viruses. METHODS: A virion-associated nucleic acids (VANA)-based metagenomics approach was used to detect mastrevirus sequences in 717 sugarcane samples from Florida (USA), Guadeloupe (French West Indies), and Réunion (Mascarene Islands). Contig assembly was performed using CAP3 and sequence searches using BLASTn and BLASTx. Mastrevirus full genomes were enriched from total DNA by rolling circle amplification, cloned and sequenced. Nucleotide and amino acid sequence identities were determined using SDT v1.2. Phylogenetic analyses were conducted using MEGA6 and PHYML3. RESULTS: We identified a new sugarcane-infecting mastrevirus in six plants sampled from germplasm collections in Florida and Guadeloupe. Full genome sequences were determined and analyzed for three virus isolates from Florida, and three from Guadeloupe. These six genomes share >88% genome-wide pairwise identity with one another and between 89 and 97% identity with a recently identified mastrevirus (KR150789) from a sugarcane plant sampled in China. Sequences similar to these were also identified in sugarcane plants in Réunion. CONCLUSIONS: As these virus isolates share <64% genome-wide identity with all other known mastreviruses, we propose classifying them within a new mastrevirus species named Sugarcane striate virus. This is the first report of sugarcane striate virus (SCStV) in the Western Hemisphere, a virus that most likely originated in Asia. The distribution, vector, and impact of SCStV on sugarcane production remains to be determined.

Genetic and morphological variants of Acidovorax avenae subsp. avenae cause red stripe of sugarcane in China.

Sugarcane (Saccharum spp.) is an important cash crop for production of sugar and bioethanol. Red stripe caused by Acidovorax avenae subsp. avenae (Aaa) is a disease that occurs in numerous sugarcane-growing regions worldwide. In this study, 17 strains of Aaa were isolated from 13 symptomatic leaf samples in China. Nine of these strains produced white-cream colonies on nutrient agar medium while the other eight produced yellow colonies. In pairwise sequence comparisons of the 16S-23S rRNA internally transcribed spacer (ITS), the 17 strains had 98.4-100% nucleotide identity among each other and 98.2-99.5% identity with the reference strain of Aaa (ATCC 19860). Three RFLP patterns based on this ITS sequence were also found among the strains of Aaa obtained in this study. Multilocus sequence typing (MLST) based on five housekeeping genes (ugpB, pilT, lepA, trpB, and gltA) revealed that the strains of Aaa from sugarcane in China and a strain of Aaa (30179) isolated from sorghum in Brazil formed a unique evolutionary subclade. Twenty-four additional strains of Aaa from sugarcane in Argentina and from other crops worldwide were distributed in two other and separate subclades, suggesting that strains of A. avenae from sugarcane are clonal populations with local specificities. Two strains of Aaa from China (CNGX08 forming white-cream colored colonies and CNGD05 forming yellow colonies) induced severe symptoms of red stripe in sugarcane varieties LC07-150 and ZZ8 but differed based on disease incidence in two separate inoculation experiments. Infected plants also exhibited increased salicylic acid (SA) content and transcript expression of gene PR-1, indicating that the SA-mediated signal pathway is involved in the response to infection by Aaa. Consequently, red stripe of sugarcane in China is caused by genetically different strains of Aaa and at least two morphological variants. The impact of these independent variations on epidemics of red stripe remains to be investigated.

Sugarcane responses to two strains of Xanthomonas albilineans differing in pathogenicity through a differential modulation of salicylic acid and reactive oxygen species.

Leaf scald caused by Xanthomonas albilineans is one of the major bacterial diseases of sugarcane that threaten the sugar industry worldwide. Pathogenic divergence among strains of X. albilineans and interactions with the sugarcane host remain largely unexplored. In this study, 40 strains of X. albilineans from China were distributed into three distinct evolutionary groups based on multilocus sequence analysis and simple sequence repeats loci markers. In pathogenicity assays, the 40 strains of X. albilineans from China were divided into three pathogenicity groups (low, medium, and high). Twenty-four hours post inoculation (hpi) of leaf scald susceptible variety GT58, leaf populations of X. albilineans strain XaCN51 (high pathogenicity group) determined by qPCR were 3-fold higher than those of strain XaCN24 (low pathogenicity group). Inoculated sugarcane plants modulated the reactive oxygen species (ROS) homoeostasis by enhancing respiratory burst oxidase homolog (ScRBOH) expression and superoxide dismutase (SOD) activity and by decreasing catalase (CAT) activity, especially after infection by X. albilineans XaCN51. Furthermore, at 24 hpi, plants infected with XaCN51 maintained a lower content of endogenous salicylic acid (SA) and a lower expression level of SA-mediated genes (ScNPR3, ScTGA4, ScPR1, and ScPR5) as compared to plants infected with XaCN24. Altogether, these data revealed that the ROS production-scavenging system and activation of the SA pathway were involved in the sugarcane defense response to an attack by X. albilineans.

Comparison of the Virome of Quarantined Sugarcane Varieties and the Virome of Grasses Growing near the Quarantine Station.

Visacane is a sugarcane quarantine station located in the South of France, far away from sugarcane growing areas. Visacane imports up to 100 sugarcane varieties per year, using safe control and confinement measures of plants and their wastes to prevent any risk of pathogen spread outside of the facilities. Viruses hosted by the imported material are either known or unknown to cause disease in cultivated sugarcane. Poaceae viruses occurring in plants surrounding the quarantine glasshouse are currently unknown. These viruses could be considered as a source of new sugarcane infections and potentially cause new sugarcane diseases in cases of confinement barrier failure. The aim of this study was to compare the plant virome inside and outside of the quarantine station to identify potential confinement failures and risks of cross infections. Leaves from quarantined sugarcane varieties and from wild Poaceae growing near the quarantine were collected and processed by a metagenomics approach based on virion-associated nucleic acids extraction and library preparation for Illumina sequencing. While viruses belonging to the same virus genus or family were identified in the sugarcane quarantine and its surroundings, no virus species was detected in both environments. Based on the data obtained in this study, no virus movement between quarantined sugarcane and nearby grassland has occurred so far, and the confinement procedures of Visacane appear to be properly implemented.

Complete Genome Sequence Reveals Evolutionary and Comparative Genomic Features of Xanthomonas albilineans Causing Sugarcane Leaf Scald.

Leaf scald (caused by Xanthomonas albilineans) is an important bacterial disease affecting sugarcane in most sugarcane growing countries, including China. High genetic diversity exists among strains of X. albilineans from diverse geographic regions. To highlight the genomic features associated with X. albilineans from China, we sequenced the complete genome of a representative strain (Xa-FJ1) of this pathogen using the PacBio and Illumina platforms. The complete genome of strain Xa-FJ1 consists of a circular chromosome of 3,724,581 bp and a plasmid of 31,536 bp. Average nucleotide identity analysis revealed that Xa-FJ1 was closest to five strains from the French West Indies and the USA, particularly to the strain GPE PC73 from Guadeloupe. Comparative genomic analysis between Xa-FJ1 and GPE PC73 revealed prophage integration, homologous recombination, transposable elements, and a clustered regulatory interspaced short palindromic repeats (CRISPR) system that were linked with 16 insertions/deletions (InDels). Ten and 82 specific genes were found in Xa-FJ1 and GPE PC73, respectively, and some of these genes were subjected to phage-related proteins, zona occludens toxin, and DNA methyltransferases. Our findings highlight intra-species genetic variability of the leaf scald pathogen and provide additional genomic resources to investigate its fitness and virulence.

Identification of Differentially Expressed Proteins in Sugarcane in Response to Infection by Xanthomonas albilineans Using iTRAQ Quantitative Proteomics.

Sugarcane can suffer severe yield losses when affected by leaf scald, a disease caused by Xanthomonas albilineans. This bacterial pathogen colonizes the vascular system of sugarcane, which can result in reduced plant growth and plant death. In order to better understand the molecular mechanisms involved in the resistance of sugarcane to leaf scald, a comparative proteomic study was performed with two sugarcane cultivars inoculated with X. albilineans: one resistant (LCP 85-384) and one susceptible (ROC20) to leaf scald. The iTRAQ (isobaric tags for relative and absolute quantification) approach at 0 and 48 h post-inoculation (hpi) was used to identify and annotate differentially expressed proteins (DEPs). A total of 4295 proteins were associated with 1099 gene ontology (GO) terms by GO analysis. Among those, 285 were DEPs during X. albilineans infection in cultivars LCP 85-384 and ROC20. One hundred seventy-two DEPs were identified in resistant cultivar LCP 85-384, and 113 of these proteins were upregulated and 59 were downregulated. One hundred ninety-two DEPs were found in susceptible cultivar ROC20 and half of these (92) were upregulated, whereas the other half corresponded to downregulated proteins. The significantly upregulated DEPs in LCP 85-384 were involved in metabolic pathways, the biosynthesis of secondary metabolites, and the phenylpropanoid biosynthesis pathway. Additionally, the expression of seven candidate genes related to photosynthesis and glycolytic pathways, plant innate immune system, glycosylation process, plant cytochrome P450, and non-specific lipid transfer protein was verified based on transcription levels in sugarcane during infection by X. albilineans. Our findings shed new light on the differential expression of proteins in sugarcane cultivars in response to infection by X. albilineans. The identification of these genes provides important information for sugarcane variety improvement programs using molecular breeding strategies.

Xanthomonas albilineans HtpG is required for biosynthesis of the antibiotic and phytotoxin albicidin.

Xanthomonas albilineans, the causal agent of leaf scald disease of sugarcane, produces a highly potent polyketide-peptide antibiotic and phytotoxin called albicidin. Previous studies established the involvement of a large cluster of genes in the biosynthesis of this toxin. We report here the sub-cloning and sequencing of an additional gene outside of the main cluster and essential for albicidin biosynthesis. This gene encodes a 634-amino-acid protein that shows high identity with the Escherichia coli heat shock protein HtpG. Complementation studies of X. albilineans Tox- mutants confirmed the requirement of htpG for albicidin biosynthesis and revealed functional interchangeability between E. coli and X. albilineans htpG genes. HtpG was co-localised with albicidin in the cellular membrane, i.e., the cellular fraction where the toxin is most probably biosynthesised. Here we show the requirement of an HtpG protein for the biosynthesis of a polyketide-peptide antibiotic.

Albicidin pathotoxin produced by Xanthomonas albilineans is encoded by three large PKS and NRPS genes present in a gene cluster also containing several putative modifying, regulatory, and resistance genes.

Xanthomonas albilineans, which causes leaf scald disease of sugarcane, produces a highly potent pathotoxin called albicidin. We report here sequencing and homology analysis of the major gene cluster, XALB1 (55,839 bp), and a second, smaller region, XALB2 (2,986 bp), involved in albicidin biosynthesis. XALB1 contains 20 open reading frames, including i) three large genes with a modular architecture characteristic of polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and ii) several putative modifying, regulatory, and resistance genes. Sequencing and complementation studies of six albicidin-defective mutants enabled us to confirm the involvement of the three PKS and NRPS genes encoded by XALB1 in albicidin production. XALB2 contains only one gene that is required for post-translational activation of PKS and NRPS enzymes, confirming the involvement of these enzymes in albicidin biosynthesis. In silico analysis of these three PKS or NRPS enzymes allowed us to propose a model for the albicidin backbone assembly and to gain insight into the structural features of this pathotoxin. This is the first description of a complete mixed PKS-NRPS gene cluster for toxin production in the genus Xanthomonas.

Substrate specificity-conferring regions of the nonribosomal peptide synthetase adenylation domains involved in albicidin pathotoxin biosynthesis are highly conserved within the species Xanthomonas albilineans.

Albicidin is a pathotoxin produced by Xanthomonas albilineans, a xylem-invading pathogen that causes leaf scald disease of sugarcane. Albicidin is synthesized by a nonribosomal pathway via modular polyketide synthase and nonribosomal peptide synthetase (NRPS) megasynthases, and NRPS adenylation (A) domains are responsible for the recognition and activation of specific amino acid substrates. DNA fragments (0.5 kb) encoding the regions responsible for the substrate specificities of six albicidin NRPS A domains from 16 strains of X. albilineans representing the known diversity of this pathogen were amplified and sequenced. Polymorphism analysis of these DNA fragments at different levels (DNA, protein, and NRPS signature) showed that these pathogenicity loci were highly conserved. The conservation of these loci most likely reflects purifying selective pressure, as revealed by a comparison with the variability of nucleotide and amino acid sequences of two housekeeping genes (atpD and efp) of X. albilineans. Nevertheless, the 16 strains of X. albilineans were differentiated into several groups by a phylogenetic analysis of the nucleotide sequences corresponding to the NRPS A domains. One of these groups was representative of the genetic diversity previously found within the pathogen by random fragment length polymorphism and amplified fragment length polymorphism analyses. This group, which differed by three single synonymous nucleotide mutations, contained only four strains of X. albilineans that were all involved in outbreaks of sugarcane leaf scald. The amount of albicidin produced in vitro in agar and liquid media varied among the 16 strains of X. albilineans. However, no relationship among the amount of albicidin produced in vitro and the pathotypes and genetic diversity of the pathogen was found. The NRPS loci contributing to the synthesis of the primary structure of albicidin apparently are not involved in the observed pathogenicity differences among strains of X. albilineans.

Heterologous production of albicidin: a promising approach to overproducing and characterizing this potent inhibitor of DNA gyrase.

The phytotoxin and polyketide antibiotic albicidin produced by Xanthomonas albilineans is a highly potent DNA gyrase inhibitor. Low yields of albicidin production have slowed studies of its chemical structure. Heterologous expression of albicidin biosynthetic genes in X. axonopodis pv. vesicatoria resulted in a sixfold increase in albicidin production, offering promising strategies for engineering overproduction.