The ins and outs of leaf scald of sugarcane

The seminar disclaimer applies to this post.

The topic of seminar for this week was the bacterial pathogen Xanthomonas albilineans, the causal agent of leaf scald of sugarcane. The speaker was Dr. Philippe Rott, from CIRAD UMR BGPI, Montpellier, France.

Sugarcane is the main source of sugar in the world. Sugarcane is propagated vegetatively from stalk cuttings. The cuttings germinate, tiller (produce new stalks), grow, and mature (final accumulation of sugar). The crop cycle is 3-10 years long. Sugarcane is grown in many countries, but Brazil, India and China are the main producers, the US is #10 on the list.

Leaf scald disease is caused by a bacterium, Xanthomonas albilineans, and the most typical symptom of leaf scald is a white pencil-line streak running parallel along the veins (Comstock and Lentini, 1991, 2005). Over time the leaf becomes necrotic and dies.
Picture credit.

Leaf scald of sugarcane was first described in 1911 in Australia, and is currently controlled to some extent by using resistant sugarcane crosses Saccharum officinarum x S. spontaneum. The genus Xanthomonas contains several major plant pathogens, including X. campestris pv. campestris (black rot of several plants), X. axonopodis pv. citri (citrus canker), X. axonopodis pv. vesicatoria (bacterial spot), and X. oryzae pv. oryzae (bacterial leaf blight of rice).

Some known pathogenicity factors (factors that the pathogen needs to cause disease) in these pathogens are the xanthan gum genes, the LPS (lipopolysaccharides that the bacteria secrete, rpf (regulation of pathogenicity factors) genes, and a type III secretion system (also called a hrp system).

X. albilineans invades the sugarcane xylem. It is an unusual xanthomonad in that is phylogenetically distinct from other plant-pathogenic xanthomonads, based on the sequences of the ITS region and gyrB. Based on PCR and Southern blot experiments, there is no evidence that X. albilineans has a hrp system like other phytopathogenic xanthomonads. The pathogen does produce a toxin, albicidin.

Albicidin is a specific molecule essential for the disease symptoms, and toxin(-) mutants show no symptoms on sugarcane. Albicidin inhibits DNA replication, chloroplasts, prokaryotes (which means it's an antibiotic), and bacteriophages.

In 1993, Dr. Rott joined the lab of Dr. Dean Gabriel in the department of Plant Pathology at the University of Florida as a visiting professor to further study X. albilineans. His goal was to characterize the biosynthetic pathway of albicidin, identify the genes involved, and study the relationship between biosynthesis variability and variability in disease symptoms.

Using Tn5 mutagenesis, he isolated 7100 mutants, 50 of which did not produce albicidin (assayed by the lack of an inhibition ring around the bacterial colonies). He identified 3 genomic regions that were involved in albicidin production. XALB1, a 55.8 kb region, a cluster of 20 predicted open reading frames, XALB2, a 2.9 kb region, predicted to encode a single protein, phosphopantheteinyl transferase (PPT), and XALB3, a 2.6 kb region, predicted to encode a single protein, a heat shock factor, HtpG (Vivien et al., 2007).

The XALB1 region contains genes that are similar to genes that encode non-ribosomal peptide synthases (NRPSs) and polyketide synthases (PKSs) in addition to predicted regulatory, modifying, and resistance genes. Mutations made in some of these genes and subsequent complementation confirmed they were necessary for albicidin synthesis (Royer et al., 2004).

NRPs and PKSs are large, multi-functional enzymes that are responsible for one specific elongation step of adding a peptide or ketide to a growing polypeptide/ketide chain. Certain domains within these proteins determine their substrate specificity (meaning they determine which particular peptide or ketide is added to the chain). The albicidin backbone is made by NRPSs and PKSs, although the complete structure has not been elucidated yet, because albicidin contains some unusual peptide residues.

Dr. Rott then set out to determine whether the diversity of the genes involved in the biosynthesis of albicidin are also involved in the pathogenicity variation observed for X. albilineans associated with different pathotypes, and disease outbreaks. Research found no structural differences in albicidin. There were differences found in the albicidin production levels, but there was no correlation with pathogenicity (Champoiseau et al., 2006). This implies that there is some other factor involved, which is responsible for the observed variation in pathogenicity. This hypothesis is supported by the fact that toxin(-) mutants are still able to colonize the sugarcane stalk, even though they do not produce symptoms.

Dr. Rott's team then set out to sequence the entire genome of X. albilineans. Because this research has not been published yet, he did not give many specifics about the sequence data. He did reveal that X. albilineans turned out even more strange than anticipated, with an unusually high number of NRPS genes, only 3 of which are required for albicidin synthesis.

Dr. Rott concluded by summarizing that X. albilineans
1) is an unusual xanthomonad that does not have high similarity in genome sequence to other published xanthomonad genomes;
2) produces no xanthan gum;
3) does not have a hrp system;
4) has a fairly small genome compared to other xanthomonads;
5) is phylogenetically distinct from other xanthomonads;
6) appears to specialize in the production of secondary metabolites, which can enter the plant cell, using NRPSs and PKSs.

References:

Champoiseau, P., Daugrois, J.-H., Girard, J.-C., Royer, M. and Rott, P.C. (2006) Variation in albicidin biosynthesis genes and in pathogenicity of Xanthomonas albilineans, the sugarcane leaf scald pathogen. Phytopathology 96(1):33-45.

Comstock, J.C. and Lentini, R.S. (1991, 2005) Sugarcane Leaf Scald Disease. Document SS-AGR-201, Agronomy Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. First printed March 1991. Revised March 2005. Online at: http://edis.ifas.ufl.edu/SC001. Last accessed: March 26, 2008.

Royer, M., Costet, L., Vivien, E., Bes, M., Cousin, A., Damais, A., Pieretti, I., Savin, A., Megessier, S., Viard, M., Frutos, R., Gabriel, D.W., and Rott, P.C. (2004) 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. Mol. Plant-Microbe Interact. 17(4)414-427.

Vivien, E., Pitorre, D., Cociancich, S., Pieretti, I, Gabriel, D.W., Rott, P.C., and Royer, M. (2007) Heterologous production of albicidin: a promising approach to overproducing and characterizing this potent inhibitor of DNA gyrase. Antimicrob Agents Chemother. 51(4): 1549–1552.