Fig 1.
Cotton bacterial blight (CBB) symptoms and reemergence across the southern United States.
(Left) Typical CBB symptoms present in cotton fields near Lubbock, TX during the 2015 growing season include angular leaf spots, boll rot, and black arm rot. Yellow shading within world map (top) indicates origin of strains included in this study. Acres of cotton planted per county in the United States in 2015 (blue) and counties with confirmed CBB in 2015 (red outline). Statistics on the area of cotton planted in the U.S. were acquired from the USDA. CBB was reported by extension agents, extension specialists, and certified crop advisers in their respective states.
Fig 2.
Phylogenetic analysis of Xcm isolates and 13 species of Xanthomonas.
A) MLST (Multi Locus Sequence Typing) and maximum likelihood analysis of 13 Illumina sequenced Xcm isolates (this paper) and 40 other Xanthomonads using concatenated sections of the gltA, lepA, lacF, gyrB, fusA and gap-1 loci. B) SNP based neighbor-joining tree generated from 17,853 variable loci between 13 Xcm isolates and the reference genome Xanthomonas citri subsp. citri strain Aw12879. The tree was made using the Simple Phylogeny tool from ClustalW2.
Table 1.
Illumina and SMRT sequenced Xcm genomes described in this paper.
Fig 3.
Molecular and phenotypic analysis of Xcm and G. hirsutum interactions.
A) Type three effector profiles of Xcm isolates were deduced from de novo, Illumina based genome assemblies. Effector presence or absence was determined based on homology to known type three effectors using the program Prokka. B) Commercial and public G. hirsutum cultivars were inoculated with 13 Xcm isolates. Susceptible (S) indicates water soaking symptoms. Resistant (R) indicates a visible hypersensitive response. Plants were screened with a range of inoculum concentration from OD600 = 0.001–0.5. C) Disease symptoms on G. hirsutum cultivars Stoneville 5288 B2F and DES 56 after inoculation with Xcm strain AR81009 (OD600 = 0.05). Symptoms are visualized under visible (VIS) and near infrared (NIR) light. D) The proportion of US fields planted with susceptible and resistant cultivars of G. hirsutum was determined using planting acreage statistics from the USDA-AMA and disease phenotypes based on previous reports for common cultivars [34–36].
Fig 4.
SMRT sequencing of two phenotypically and geographically diverse Xcm isolates: MS14003 and AR81009.
Circos plot visualization of two circular Xcm genomes. Tracks are as follows from inside to outside: synteny of gene models; GC Content; DNA Methylation on + and–strands; location of type three effectors (teal) and TAL effectors (red), and position. On each side, accompanying plasmids are cartooned. Type three effector repertoires and the type IV secretion systems were annotated using Prokka. Homologous regions greater than 1kb were identified using MAUVE, and TAL effectors were annotated using AnnoTALE.
Fig 5.
SMRT sequencing and western blot reveal diverse TAL effector repertoires between Xcm strains MS14003 and AR81009.
Western Blot of TAL effectors using polyclonal TAL-specific antibody and gene models of TAL effectors identified by AnnoTALE. Blue and Green highlighted gene models represent TALs grouped in the same clade by repeat variable di-residue (RVD) sequence using AnnoTALE.
Fig 6.
RNA-sequencing analysis of infected G. hirsutum tissue demonstrates transcriptional changes during CBB.
A) Disease phenotypes of Xcm strains MS14003 and AR81009 on G. hirsutum cultivars Acala Maxxa and DES 56, 7 days post inoculation. B) Acala Maxxa and DES 56 were inoculated with Xcm strains MS14003 and AR81009 at an OD of 0.5 and a mock treatment of 10mM MgCl2. Inoculated leaf tissue was collected at 24 and 48 hpi (before disease symptoms emerged). C) Venn diagram of upregulated G. hirsutum genes (Log2(fold change in FPKM) ≥ 2 and p value ≤ 0.05) in response to Xcm inoculation. Venn diagram was created using the VennDiagram package in R.
Fig 7.
Expression of homeologous pairs across the A and D G. hirsutum genomes in response to Xcm inoculation.
Genes are considered up or down regulated if the absolute value of gene expression change after inoculation as compared to mock treatment was Log2(fold change in FPKM) ≥ 2 and p value ≤ 0.05. By these criteria, pink shading indicates no significant gene expression change. A-D-: both members of the homeologous gene pair are down regulated; A-D0: only the ‘A’ sub-genome homeolog is down regulated; A-D+: ‘A’ sub-genome homeolog is down regulated, ‘D’ sub-genome homeolog is upregulated; etc. Number of gene pairs (n) meeting each expression pattern is indicated within the grey bar. For all genes meeting each expression pattern, the distribution of expression patterns is displayed as a box plot. Rectangles indicate the interquartile range and the whiskers show 1.5 times the interquartile range. A) Acala Maxxa inoculated with MS14003 B) DES 56 inoculated with MS14003 C) Acala Maxxa inoculated with AR 81009 D) DES 56 inoculated with AR81009.
Table 2.
Eight homeologous pairs of Gossypium hirsutum genes are upregulated in both Acala Maxxa and DES 56 varieties 48 hours post inoculation with Xanthomonas citri pv. malvacearum strains MS14003 and AR81009.
Fig 8.
Three candidate G. hirsutum susceptibility genes are targeted by two different Xcm strains.
A) The homeologous pair of SWEET genes A04_G0861 and D04_G1360 are upregulated in the presence of Xcm strain MS14003. (top) Cartoon summary of 300bp promoters of A04_G0861 and D04_G1360. (bottom) Heat-map of the expressions of A04_G0861 and D04_G1360 48 hours after mock or Xcm inoculation. B) The SWEET gene D12_G1898 is upregulated in the presence of Xcm strain AR81009. (top) Cartoon summary of 300bp promoters of D12_G1898 and A12_G1747. (bottom) Heat-map of the expressions of A12_G1747 and D12_G1898 48 hours after mock or Xcm inoculation. TAL effector binding sites were predicted with TALEsf using a quality score cutoff of 4. Gene promoter cartoon legend: Arrow: TAL effector binding site; Black dot: Deletion; Black bar: SNP; Pink bar: TATA box; Teal section: 5’UTR.