Three Xanthomonas Cell Wall Degrading Enzymes and Sorghum Brown midrib12 Contribute to Virulence and Resistance in the Bacterial Leaf Streak Pathosystem

Sorghum bicolor (L.) Moench is an important grass species closely related to maize (Zea mays L.), and sugarcane (Saccharum spp.) (Swigoňová et al. 2004) and is used to produce biomass for fodder biofuels as well as grain for human consumption and animal feed. Improving the digestibility of cell walls and increasing the endogenous sugar content of bioenergy crops like sorghum is a major research focus (Vermerris 2011). At the same time, an ideal bioenergy crop will thrive on marginal lands and display robust biotic stress tolerance. Sugar profiles and cell wall characteristics are highly relevant to plant-pathogen interactions. Therefore, this menu of desired bioenergy traits likely poses some challenging tradeoffs.

Plant cell walls are essential, complex matrices of carbohydrates and phenolic compounds. To function, the composition of the plant cell wall has evolved to be sufficiently rigid yet flexible, providing structural support and facilitating water transport while enabling growth and preventing breakage (Höfte and Voxeur 2017). The primary structural component of cell walls is broadly referred to as lignocellulose, which depending on the species and tissue, consists of variable amounts of the cellulosic, hemicellulosic, and pectic polysaccharides, as well as the aromatic polymer lignin. Lignin is present in plant secondary cell walls, where it facilitates transport of water through xylem and provides structural support to sclerenchyma cells. Lignin is formed by the plant through the oxidative polymerization of monolignols (Ralph et al. 2019; Vanholme et al. 2019). Often referred to as the most abundant renewable resource on the planet, lignocellulose comprises the vast majority of plant biomass and is of particular interest as a renewable, sustainable energy source (Kerr 2007; Liu et al. 2021). Improved digestibility of recalcitrant lignocellulose is achieved by directly reducing lignin content, modifying the lignin subunit composition and introducing labile chemical bonds into the lignin that can be broken under mild conditions (Poovaiah et al. 2014; Vermerris and Abril 2015). Mutants affected in phenolic metabolism are useful for the identification of compounds and mechanisms involved in both defense responses and cell wall formation. The brown midrib (bmr) mutants of sorghum are of particular interest in these respects. These mutants were first identified among a population of chemically mutagenized plants (Bittinger et al. 1981; Porter et al. 1978), and subsequent bmr mutants have been reported (Xin et al. 2009). Allelism tests have established a total of eight independent loci among the collections of bmr mutants identified to date (Saballos et al. 2008; Sattler et al. 2014). The Bmr12, Bmr6, and Bmr2 genes encode the enzyme caffeic acid O-methyltransferase (SbCOMT) (Bout and Vermerris 2003), cinnamyl alcohol dehydrogenase (SbCAD2) (Saballos et al. 2009; Sattler et al. 2009), and 4-coumaric acid CoA ligase (Sb4CL) (Saballos et al. 2012), respectively. Recently, the Bmr19 gene was shown to encode folylpolyglutamate synthase (Adeyanju et al. 2021), and the Bmr30 gene encodes chalcone isomerase (Tetreault et al. 2021), which is involved in the biosynthesis of tricin, a flavonoid that serves as nucleation site for lignin in grasses (del Río et al. 2012; Lan et al. 2015). Consequently, bmr mutants contain less lignin and/or have altered lignin subunit composition (Bucholtz et al. 1980; Pillonel et al. 1991; Porter et al. 1978). Thermochemical pretreatment followed by enzymatic hydrolysis of cell wall polysaccharides of biomass from the bmr6 and bmr12 mutants has been shown to increase the yield of fermentable sugars (Dien et al. 2009; Saballos et al. 2008; Vermerris et al. 2007).

The plant cell wall is an enticing target for improving biofuel production. However, it is also critical for protection against pathogens, serving as a physical barrier to pathogen entry and spread (Ishida and Noutoshi 2022; Underwood 2012). Successful pathogens enter plant leaves through stomata, hydathodes, or wounds. In the apoplast, many bacterial pathogens produce plant cell wall degrading enzymes (CWDEs) that target specific components like cellulose, xylan, and pectins (Esquerré-Tugayé et al. 2000; Pinski et al. 2019; Tom et al. 2022). Production of CWDEs facilitates pathogen access to plant cell plasma membranes and movement through the plant (Cantu et al. 2008; Doi and Kosugi 2004; Rajeshwari et al. 2005). CWDEs, including cellulases and pectinases, are secreted through the Type-II Secretion System (T2SS) (da Silva et al. 2002; Jacques et al. 2016; Lu et al. 2008; Potnis et al. 2011). Several types of CWDE have indeed been found to be important for full pathogen virulence (Meena et al. 2019), including cellulolytic enzymes of Ralstonia solanacearum (Liu et al. 2005) and pectate lyases from Dickeya dadantii (formerly known as Erwinia chrysanthemi) (Franza et al. 1999; Hugouvieux-Cotte-Pattat et al. 1996), both of which cause bacterial diseases in a wide range of ornamental and horticulturally important host plants.

Xanthomonads are bacterial pathogens that cause disease in over 400 diverse plant species, including many important crops like sorghum (Wang et al. 2021), rice (Oryza sativa) (Zhang and Wang 2013), cassava (Manihot esculenta) (Bart et al. 2012; Cohn et al. 2014; Zárate-Chaves et al. 2021), cotton (Gossypium hirsutum) (Phillips et al. 2017), cabbage (Brassica oleracea) (Pandey et al. 2017), beans (Phaseolus vulgaris) (Darsonval et al. 2008), tomato (Solanum lycopersicum), pepper (Capsicum annuum) (Potnis et al. 2011), and many members of the genus Citrus (Ference et al. 2018). Sorghum bacterial leaf streak disease, caused by Xanthomonas vasicola pv. holcicola (Xvh), has a wide geographical distribution but has historically only caused minor crop losses (Borkar and Yumlembam 2016). However, under environmental conditions favorable to the pathogen, this disease can cause considerable damage in sorghum (Claflin et al. 1992; Hartman et al. 2020; Stack 2003). The factors that cause elevation of disease severity are not well understood, and no disease management protocols are established. With the prospect of changing environmental conditions resulting from climate change and the increasing demand for lignocellulosic biomass as feedstock for renewable fuels and chemicals, it is important to have a better understanding of the interactions between sorghum and Xvh.

In summary, the plant cell wall is essential for pathogen protection and is a prime target for modification to facilitate biofuel production. How might the latter affect the former? To address this potential tradeoff, we first analyzed our previously published dataset (Wang et al. 2021) of dual RNA-seq on Xvh-infected sorghum across distinct, observable disease phenotypes. Many Xvh genes encoding CWDEs were highly induced in planta, and mutation of any of three cellulases (GH5, Bgl, and Cbh) decreased the virulence of the mutants. In the host, the sorghum Bmr12 gene was strongly induced in the tolerant sorghum genotype NTJ2 after Xvh infection but was only slightly induced in the susceptible variety, Black Spanish (BS). We show that resistant sorghum variety BTx631 is more susceptible to Xvh following introgression of the bmr12-ref mutant allele. Bmr12 encodes caffeic acid O-methyltransferase (COMT), which generates sinapaldehyde during monolignol biosynthesis (Green et al. 2014). Assays performed on Xvh growth in vitro demonstrate that sinapaldehyde has antibacterial properties. These results show that Xvh CWDEs contribute to bacterial virulence and that sorghum varieties harboring the bmr12 mutation are at risk for increased susceptibility to this bacterial pathogen.

Results

RNA-seq and biochemical analyses indicate that sorghum carbon partitioning and metabolism remain at steady state during Xvh infection

We previously reported that diverse sorghum genotypes show three different phenotypes after challenge with the bacterial pathogen, Xvh: water-soaked lesions, red lesions, and tolerance/resistance (hereafter referred to as tolerance). For each disease phenotype class, we selected one representative variety (BS: water-soaked lesions; Grassl: red lesions; and NTJ2: tolerance) and characterized the transcriptional responses, in plant and bacteria, that occur after inoculation. In that previous work, we focused on type three effectors and pathogenesis-related (PR) genes in the bacteria and plant, respectively (Wang et al. 2021). Here, we turn our attention to other features of the pathogen virulence arsenal and plant defense systems. It is well established that many pathogens actively alter carbon partitioning and metabolism and/or cell wall characteristics during host-pathogen interactions (Chen et al. 2010; Esquerré-Tugayé et al. 2000; Kanwar and Jha 2019; Vogel et al. 2002), although this has not been characterized in the sorghum-Xvh pathosystem. First, we analyzed the expression patterns of known sugar related genes (sucrose, glucose, and fructose) and observed that the majority of these genes were not responsive to pathogen infection in any of the three sorghum genotypes (Supplementary Fig. S1). A single putative glucose transporter gene (Sorghum bicolor v3.1.1 locus ID: Sobic.002G201900) showed induction after Xvh infection in all three sorghum varieties. As a complementary approach, we directly compared soluble sugar and starch levels in mock- or Xvh-infiltrated leaves during susceptible and tolerant interactions. We did not observe an increase in sugars, including glucose, or starch levels upon pathogen infection (Supplementary Fig. S1). However, after pathogen infection, the most susceptible genotype, BS, contained decreased sucrose and fructose, and both BS and Grassl showed decreased starch. Next, we considered the features of the cell wall. The transcriptome data indicated that many Xvh cellulolytic enzymes were more strongly induced in planta than in vitro (Supplementary Fig. S2). Given these observations, we hypothesized that bacterial CWDEs may contribute to the virulence of Xvh.

Expression of Xvh CWDEs during sorghum infection is required for full virulence of the pathogen

We selected the three CWDEs that were consistently and highly induced during infection: a member of glycosyl hydrolase family 5 (GH5, gene ID 2758111359, E.C. 3.2.1.4), a beta-glucosidase (Bgl, gene ID 2758111888, E.C. 3.2.1.21), and a 1,4-beta-cellobiosidase (Cbh, gene ID 2758111991, E.C. 3.2.1.91) (Fig. 1). We generated deletion mutants for these genes encoding the putative CWDEs (XvhΔGH5, XvhΔBgl, and XvhΔCbh) in the wild-type (WT) background of Xvh and determined that there were no differences in bacterial growth in vitro relative to WT (Fig. 2A to D). We tested the ability of each mutant compared with a complemented version of the strain, to degrade carboxymethyl cellulose (CMC), and observed that these mutants had lower CMC degradation abilities compared with the WT Xvh strain in vitro (Fig. 2E). To test whether these CWDEs contribute to the virulence of Xvh in plants, we compared the disease phenotype and bacterial growth of WT Xvh and the CWDE mutants in the susceptible sorghum variety BS. The CWDE mutants caused dark, water-soaking phenotypes, similar to WT Xvh, in infected plants (Fig. 3A). However, the CWDE knockout mutants displayed reduced bacterial growth compared with WT Xvh (Fig. 3B). These data indicate that Xvh CWDEs contribute to virulence during sorghum bacterial leaf streak infection.

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Cell wall compositional analyses do not reveal changes in response to infection

Plant cell walls are based on a co-extensive load-bearing network of cellulose microfibrils cross-linked with hemicelluloses and are reinforced by the deposition of lignin. As lignification of cell walls is a known defense mechanism employed by plants (Lee et al. 2019), we tested to see whether we could detect differences in the cell wall composition in resistant (NTJ2) and susceptible (BS) sorghum both before and after infection. Comparisons were made between inoculated and mock-inoculated samples of the resistant genotype NTJ2 and the susceptible genotype BS. We hypothesized that inherent genotypic variation in cell wall composition could explain the difference in the response to infection with Xvh and/or that the resistant genotype would mount a defense response involving a modification of the cell wall, for example by depositing hydroxycinnamic acids or additional lignin, possibly with a different subunit composition than the native lignin. Initial attempts to analyze variation in cell wall composition in response to infection in 14-day-old plants revealed there was too little lignin for meaningful analyses (data not shown). Instead, leaf tissue of 60-day-old greenhouse-grown plants was inoculated with Xvh or a mock inoculum, and leaf tissue was collected at 9 days postinfection (dpi). Midrib tissue was dissected and analyzed using quantitative pyrolysis-GC-MS to obtain insight into lignin content and structural features.

The analysis revealed that the lignin concentration in NTJ2 midribs was 1.4-fold higher than in BS midribs (Supplementary Fig. S3) but that the infection did not result in changes in lignin concentration in either genotype. In other words, there was no evidence for the deposition of additional lignin in response to infection. Nonetheless, the higher lignin content of the NTJ2 genotype could point towards a role in its tolerance. The lignin subunit composition, expressed as the ratio of syringyl to guaiacyl residues (S/G ratio) was similar in both genotypes and did not change in response to infection (Supplementary Table S4). The cell walls of resistant genotype NTJ2 contained less p-coumarate (identified in the pyrograms as 4-vinylphenol) than observed in BS (Supplementary Table S4). Because pyrolysis of aromatic amino acids also results in p-hydroxyphenyl products, including 4-vinylphenol, we monitored the abundance of indole as a pyrolysis marker of tryptophan. From the identical indole abundances in both genotypes, we inferred that the observed difference in 4-vinylphenol could indeed be ascribed to p-coumarate. This hydroxycinnamic acid might be esterified to glucuronoarabinoxylans (GAX), like ferulate, but is mainly found acylating the Cγ-position of syringyl residues in the lignin (del Río et al. 2008, 2015; Ralph et al. 1994). Given the lack of variation in lignin subunit composition between the two genotypes (Supplementary Table S4), this additional p-coumarate might be associated with GAX. There were, however, no changes in the amount of p-coumarate present in infected versus mock-inoculated midribs. Analysis of the composition of cell wall polysaccharide-derived pyrolysis products did not reveal substantial differences among genotypes and infection status either (Supplementary Table S5). Taken together, the higher concentration of lignin in NTJ2 and the smaller amount of p-coumarate esterified to GAX may contribute to the resistance of NTJ2 by making the cell wall more recalcitrant to CWDEs secreted by Xvh. Our data reveal no evidence for a dynamic change in cell wall composition in response to infection in either genotype.

Monolignol biosynthetic enzymes from sorghum contribute to sorghum resistance to bacterial leaf streak infection

The Bmr genes are involved in phenolic metabolism, defense responses, and lignin formation. Bmr6 and Bmr12 are both involved in monolignol biosynthesis in sorghum (Supplementary Fig. S4), so we re-analyzed the transcript levels of Bmr6 and Bmr12 from our previous study (Wang et al. 2021). The expression level of Bmr6 was relatively low (fragments per kilobase of transcript per million mapped reads [FPKM] value ≤ 15) in all three sorghum genotypes analyzed. However, expression of Bmr12 was strongly upregulated in the resistant interactions (Fig. 4). In contrast, induction of Bmr12 was relatively weak in the susceptible variety, BS. These results suggest that Bmr12, not Bmr6, may contribute to sorghum resistance to Xvh. To further test this hypothesis, we obtained the resistant genotype BTx631 and two near-isogenic lines homozygous for either the previously described bmr6-ref (Saballos et al. 2009) or bmr12-ref mutant alleles (Bout and Vermerris 2003; Pedersen et al. 2006; Fig. 5A). Infection assays with Xvh showed that bmr12 mutant plants displayed a distinct water-soaked phenotype at 7 dpi, whereas WT BTx631 and the bmr6 mutant appeared qualitatively resistant (Fig. 5B). Additionally, the bmr12 mutant plants contained significantly higher bacterial populations (Fig. 5C). In contrast, the bmr6 mutant displayed symptoms and bacterial populations similar to the WT. These results indicate that upregulation of the Bmr12 gene can contribute to resistance to Xvh in sorghum. Taken together, these data support the hypothesis that disruption of genes important for cell wall biosynthesis may simultaneously compromise disease resistance in plants.

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A product of Bmr12 enzymatic activity, sinapaldehyde, has antibacterial properties

Many phenolic intermediates are produced during the biosynthesis of monolignols (Supplementary Fig. S4). The expression of Bmr12 was highly induced in the resistant sorghum genotype NTJ2 by Xvh infection but not in the susceptible genotype BS (Fig. 4). Given that bmr12 mutants also show reduced resistance to Xvh infection (Fig. 5), we hypothesized that a reduction in sinapaldehyde production, one of the products of COMT (Supplementary Fig. S4), may contribute to bmr12 mutant susceptibility. Importantly, the upregulation of the Bmr12 gene did not result in a change in lignin subunit composition upon infection (Supplementary Table S4), as elaborated upon above, which implies that sinapaldehyde concentrations could have increased. To test the bacteriostatic activity of sinapaldehyde over time, we characterized the Xvh populations in growth media containing different concentrations of sinapaldehyde. The results showed that sinapaldehyde was able to significantly inhibit bacterial growth at a concentration equal to or greater than 4 mM at the 8-h time point (Fig. 6A). Additionally, nondetectable (ND) levels of bacteria were observed at 6 and 12 mM concentrations after 12 h and in 4 mM concentrations after 24 h. In addition, sinapaldehyde possessed a mild bactericidal activity, causing bacterial cell death of Xvh in vitro, as evidenced by a reduction of colony forming units (CFU) in plating assays (Fig. 6B). Together, our data indicate that sinapaldehyde can have antibacterial activity in vitro and present a possible chemical mechanism by which Bmr12 could contribute to host defense against Xvh.

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Discussion

Realizing the potential of plant-derived energy sources necessitates the development of high biomass-yielding crops that are amenable to processing. The ability to efficiently extract and utilize the energy stored in lignocellulosic biomass is a well-defined technological bottleneck, as these structural polymers are resistant to degradation and processing for biofuel production (Broda et al. 2022; Chang 2007; Lewis 1988). One strategy to address this is the development of specialized crop varieties that harbor modifications to cell wall structures and store high levels of nonstructural carbohydrates and hence display altered carbon partitioning. Given the importance of the cell wall in plant immunity, these modifications have the potential to present previously unseen challenges for disease resistance. However, widespread planting of sorghum varieties with altered cell wall composition or increased sugar content may have unintended consequences for plant defense.

A major bottle neck for plant scientists is functional characterization of genes. This is especially true with large gene families; finding which member(s) is critical for a specific process can be a daunting task. Transcriptome sequencing has been proposed as an efficient and economical strategy for prioritizing gene knockout studies (Wang et al. 2009). Correspondingly, we analyzed our previously published dataset (Wang et al. 2021) of dual RNA-seq on Xvh and infected sorghum. Out of 12 predicted cellulolytic enzymes, RNA-seq analysis pinpointed three that were highly induced 48 h after infection. Mutation of each of these three genes, encoding the cellulases GH5, Bgl, and Cbh, resulted in decreased activity in a carboxymethyl cellulase (CMCase) activity assay, and in each case, complementation was observed through the CMCase assay. However, we note that in the case of Bgl, the complemented strain had a unique colony morphology, and in a couple of the experimental replicates, full complementation was not observed. Complementation was done by introducing a WT version of the gene, including approximately 1,000 bp upstream of the coding sequence, into the mutant strain on the pVSP61 plasmid. The variability observed for the complemented Bgl mutant strain might indicate that the 1,000-bp promoter region used in the complementation construct contains unknown regulatory elements. GH5, Bgl, and Cbh each have unique Enzyme Commission (EC) classifications as predicted by Expasy, suggesting potentially distinct functions, in which case a triple mutant might show a stronger phenotype. CWDEs are secreted through the type II secretion system (Chang et al. 2014; Cianciotto and White 2017; Sun et al. 2005). We looked for secretion signals using SignalP and saw high confidence signals for GH5 and Cbh but not Bgl. This may be a limitation of computational prediction tools or additional support for unique functions among the three enzymes. Regardless, as predicted, each mutation decreased the virulence of Xvh in planta while not negatively affecting bacterial growth in vitro. The Xvh cellulase Cbh was induced approximately 10× more highly than either GH5 or Bgl in planta versus in culture (Fig. 1). However, XvhΔCbh was not measurably more hindered in its ability to cause disease in sorghum than the other knockouts (Fig. 3). Taken together, our data demonstrate the importance of the more laborious knockout- and infection-based experiments to further understand gene function and relevance at a biological level and suggest that Bgl, in particular, might warrant further investigation.

On the plant side, RNA-seq analysis was useful for pinpointing key genes. We observed that the sorghum Bmr12, but not Bmr6, gene was strongly induced in the resistant sorghum genotype NTJ2 after Xvh infection. Like the CWDE example, the RNA-seq analysis led us to biologically relevant genes. In this case, we showed that mutations in bmr12 in the resistant sorghum inbred BTx631 caused an increase in susceptibility, as measured by the presence of water-soaking symptoms and increased bacterial growth in infected plants. Thus, while RNA-seq analysis clearly has limitations, it is a powerful tool for prioritizing research and generating new hypotheses and is an ideal complement to genetic experiments.

Lignification can play an important role in a host's defense against pathogen invasion. As a physical barrier encountered by most plant pathogens, lignin can be rapidly deposited in cell walls in response to pathogen attack (Lee et al. 2019). Analysis of younger plant tissue was inconclusive due to overall low levels of lignin. Our analyses of 60-day-old plants revealed a higher lignin content and implied a lower level of p-coumaroylation of GAX in the resistant genotype NTJ2 compared with the susceptible genotype BS. The higher lignin content may have provided a level of protection against the pathogen even though no dynamic changes in cell wall composition were identified in response to infection in either NTJ2 or BS. Based on the transcriptome and functional characterization of cell wall-related genes in both plant and pathogen, cell wall characteristics are clearly important in this pathosystem. In the case of the plant, the data presented suggest that pathogen defense in the context of the lignin biosynthetic pathway may be tied more to byproducts of this pathway, as opposed to active reinforcing of the cell wall. COMT, the enzyme encoded by Bmr12, is responsible for generating sinapaldehyde and sinapyl alcohol, which form syringyl (S) residues following oxidative polymerization with the growing lignin polymer (Supplementary Fig. S4). Given the demonstration here that a functional Bmr12 gene was necessary for full resistance in BTx631 and the lack of evidence for enhanced lignification or change in subunit composition in response to infection, the compound(s) produced by a functional Bmr12 may instead have antimicrobial activity. In vitro assays performed as part of this work showed that one such compound, sinapaldehyde, was able to inhibit the growth of Xvh, suggesting a possible antibacterial mechanism.

Sorghum bmr12 mutants have attracted interest as feedstocks for bioenergy production because of their enhanced processing characteristics (Cotton et al. 2013; Sattler et al. 2012; Vermerris et al. 2007). Even though bmr12 mutants displayed enhanced resistance to infection with the fungal pathogens Fusarium and Alternaria (Funnell and Pedersen 2006; Funnell-Harris et al. 2010), the results from our study indicate that bmr12 sorghum may be at greater risk for Xvh infection. Similarly, modifications to the plant cell wall may have other unintended consequences. For example, recent work in rice has indicated that alterations of cell wall components can affect the larger plant microbiome (Su et al. 2024). Taken together, this work presents a strategy for using transcriptomic sequencing to pinpoint key features of an understudied pathosystem and highlights how alterations for one outcome may have unintended consequences in other areas.

Materials and Methods

Plant material and growth conditions

Sorghum genotypes BS, Grassl, and NTJ2 were used as previously described (Wang et al. 2021). WT sorghum genotype BTx631 (PI 552861) and bmr mutants (BTx631-bmr6 [PI 639717 B] and BTx631-bmr12 [PI 639718 B]) have been previously described (Pedersen et al. 2006) and were obtained from the Germplasm Resources Information Network (GRIN; Griffin, GA, U.S.A.). Indicated sorghum genotypes were grown in pots in a growth chamber set at 28/23°C with 14/10 h (day/night) conditions and 50% relative humidity. Fourteen-day-old plants were used for all experiments except cell wall compositional analyses.

Bacteria strain preparation and bacterial mutant generation

Xvh BLS185 (Xvh) was used in this study (GenBank: SMGF00000000.1). For generating the Xvh mutants, an allelic exchange strategy (Hmelo et al. 2015) was employed with some modifications. In brief, two sequence fragments for the upstream and downstream homologous regions of the target gene were cloned into the sucrose counter-selection allelic exchange vector pDEST2T18ms by Gateway technology (pDEST2T18ms was a gift from Brian Kvitko [Addgene plasmid number 72647; http://n2t.net/addgene:72647; RRID:Addgene_72647]). The recombinant plasmids were verified by Sanger sequencing and subsequently transferred into the WT Xvh strain via electroporation. Xvh cells were made competent utilizing the following microcentrifuge-based procedure: 6 ml of an overnight culture (OD_600nm = 0.05 – 0.1) grown in NYGB medium was equally distributed into four microcentrifuge tubes, and the cells were harvested by centrifugation at room temperature for 1 to 2 min at 16,000 × g. The cell pellet in each tube was washed twice with 1 ml of room temperature 300 mM sucrose, and the four cell pellets were then resuspended in a combined total of 100 μl of sucrose (300 mM), which contained on average of 10⁹ to 10¹⁰ viable bacteria; 2 μg of plasmid was added to 100 μl of electrocompetent cells and was mixed; mixtures were placed in a pre-chilled, sterile, 2-mm electroporation cuvette. The cells were electroporated with a BioRad Gene Pulser II electroporation system (Bio-Rad Laboratories) at 2.5 kV, 25 μF, and 50 Ω; it was added to 1 ml of NYG medium (per liter: 5.0 g of peptone, 3.0 g of yeast extract, and 20.0 g of glycerol); and it was incubated at 30°C for 3 h. Cells were then plated on solid NYG medium (1% [wt/vol] agar) supplemented with tetracycline (5 µg/µl). Resistant clones were selected. The mutants were further characterized by directly sequencing the PCR product. The primers are listed in Supplementary Table S1. To complement the CWDE mutants, the WT locus of Bgl, Cbh, and GH5, along with approximately 500 bp of their respective native promoters, were cloned into pVSP61 by infusion (Takara) cloning. Plasmids were confirmed to have full-length genes without errors by sequencing. Plasmids were then introduced into competent Xvh via electroporation.

Dual RNA-seq transcriptomics analysis of infected plants

RNA extraction, library generation, sequencing, and gene expression analysis was performed as a part of a previously published study (Nobori et al. 2018; Wang et al. 2021). Briefly, plant and bacterial RNA was extracted from needleless syringe-inoculated plants containing approximately 1 × 10⁸ WT Xvh cells suspended in 10 mM MgCl₂ 48 h postinoculation (hpi). Samples were DNase-treated, and ribosomal RNA was removed. cDNA libraries for Illumina sequencing were generated, and HiSeq platforms with paired-end 150-bp sequencing strategies were used for sequencing by Novogene (Sacramento, CA, U.S.A.). Reads were trimmed and aligned against a concatenated genome composed of the sorghum nuclear genome (version Sorghum bicolor v3.1.1; https://phytozome-next.jgi.doe.gov/), the sorghum chloroplast genome (GenBank: EF115542.1), the sorghum mitochondrial genome (NCBI Reference Sequence: NC_008360.1), and the Xvh genome. The draft genome of Xvh is also previously described (Wang et al. 2021) and is publicly available (GenBank: SMGF00000000.1). StringTie (version 1.3.5; Pertea et al. 2015) was used to perform quantification, generating FPKM values. The dendrogram was created using base R functions dist and hclust from the stats package (v3.4.4), which uses Euclidean distance and complete agglomeration, respectively. The z-transformed FPKM for each condition is shown with the color gradient. The RNA-seq data has been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus database (accession number GSE142035). Putative EC numbers were predicted using Expasy (https://www.expasy.org/; accessed 10 November 2024).

Bacterial infection and growth assay

Indicated Xvh strains were grown on plates containing NYGA medium for 48 h at 30°C. Next, bacteria were scraped from the plate and suspended in sterile 10 mM MgCl₂ to an OD_600nm of 0.02 (approximately 10⁷ CFU/ml). The fourth leaf of 14-day-old sorghum plants was inoculated with either bacterial suspensions or the mock treatment (10 mM MgCl₂) using a needleless syringe. The inoculation was made between 1:00 to 3:00 PM. The infected tissue areas were harvested at either 0 or 48 hpi. At each time point, one sorghum leaf disc (3-mm diameter) was cut around the inoculation point using a cork-borer. Two leaf disc sections from two inoculation areas in one leaf were combined per replicate. Replicate sizes are indicated in each figure legend. Samples were ground using the Qiagen TissueLyser (2 min, at 30 Hz) in 10 mM MgCl₂ with a 3-mm glass bead in a 2.0-ml Eppendorf Safe-Lock tube. Serial dilutions were plated on NYGA medium with appropriate selection plus cycloheximide (100 µg/µl) to inhibit fungal growth. Log₁₀-transformed colony forming units per square centimeter of leaf surface area were calculated to estimate bacterial populations.

Sugars and starch quantification

Sucrose, glucose, fructose, and starch were quantified from 100 mg of frozen sorghum leaf tissue 7 dpi after either mock or Xvh infection. Soluble sugar and starch samples were extracted as described (Leach and Braun 2016) and quantified using high-performance anion exchange chromatography against known standards (Leach et al. 2017).

Cell wall compositional analyses with pyrolysis-gas chromatography-mass spectrometry (GC-MS)

Pyrolysis-GC-MS is an analytical technique that relies on the thermal degradation of samples in an anoxic environment. In the case of plant cell wall samples, fragments are generated that are diagnostic of the main constituents of the cell wall, namely cellulose, hemicellulosic polysaccharides, hydroxycinnamic acids, and lignin (Mulder et al. 1992; Ralph and Hatfield 1991). The addition of ¹³C-labeled lignin to plant samples serves as an internal standard that enables quantitative analysis of lignin content and lignin structural features (van Erven et al. 2017, 2019).

Sixty-day-old greenhouse-grown plants of genotypes NTJ2 (resistant) and BS (susceptible) were inoculated with Xvh or mock inoculated, as described above. The area of infiltration was indicated with a black marker. Leaf tissue from three independent biological replicates was collected at 9 dpi and dried at 40°C. Midrib tissue from the inoculated area was dissected with a scalpel and crushed. Samples were analyzed for lignin content and structural features by quantitative pyrolysis-GC-MS, as previously described (van Erven et al. 2019). Uniformly ¹³C-labeled lignin (97.7 atom% ¹³C), isolated from ¹³C wheat straw (IsoLife BV, Wageningen, The Netherlands) was used as an internal standard (¹³C lignin IS) (van Erven et al. 2017). To each accurately weighed sample (132 ± 18 µg) (XP6 Excellence-Plus microbalance, Mettler-Toledo International Inc., Columbus, OH, U.S.A.), 10 μl of a 1.0 mg ml⁻¹ solution of ¹³C lignin IS dissolved in 50:50 (vol/vol) ethanol/chloroform was added. The samples were dried at 30°C for 3 h. All samples were prepared and analyzed in triplicate. Lignin-derived pyrolysis products were monitored in full MS mode on the most abundant fragment per compound (both nonlabelled and uniformly ¹³C labeled) (Supplementary Table S2). Pyrograms were processed by TraceFinder 4.0 software. Lignin contents and relative abundances of lignin-derived pyrolysis products were calculated as described previously (van Erven et al. 2019). Lignin contents were also conservatively calculated considering potential interference by ferulic acid by excluding 4-vinylguaiacol and potential interference by aromatic amino acids by excluding p-hydroxyphenyl products (H-units). The tryptophan pyrolysis marker indole (R.T. 17.10 min, m/z 117.05733) and various carbohydrate-derived pyrolysis products (Supplementary Table S3) were also monitored in full MS mode on the most abundant fragment per compound but conversely to lignin without applying corrections for (relative) response nor internal standard behavior.

CWDE activity assay

CMCase activity was measured using a protocol modified from (Sun et al. 2005). Xanthomonas cultures were grown for 48 h in NYGB, pelleted, washed with Xanthomonas minimal media (Liu et al. 2013), and resuspended in Xanthomonas minimal media to an OD₆₀₀ nm of 1.0. After incubation for approximately 4 h, 10 µl was spotted on NYGA plates (2% [wt/vol] agar) containing 0.5% (wt/vol) carboxymethyl cellulose (CMC; 265 Sigma). Plates were sealed with Parafilm and incubated for 2 days at 30°C. The colony spread was marked, and the diameter was measured in two places. Petri dishes were then flooded with an aqueous solution of Congo Red dye (1% [wt/vol] Congo Red in distilled water) and shaken at 60 rpm for 15 min. The Congo red solution was then poured off, and plates were rinsed with 1M NaCl. The plates were then flooded with 1M NaCl and shaken at 60 rpm for 15 min. The cellulase activity of the strains was detected by the formation of a transparent halo against the red background after staining with Congo Red. The enzymatic activity index was calculated as the width of the clear halo plus the colony diameter divided by the diameter of the colony.

Antibacterial activity of sinapaldehyde

The antibacterial activity of sinapaldehyde on Xvh growth was measured across time and at different concentrations. First, bacterial populations were determined across multiple time points under treatment with different concentrations of sinapaldehyde or eugenol to determine bacteriostatic activity. The initial population WT Xvh used was OD_600nm = 0.02 (approximately 1 × 10⁷ CFU/ml), and samples were then taken and plated for determining viable colony forming units per milliliter of bacterial culture at each time point. Additionally, bactericidal activity of sinapaldehyde on Xvh was determined. Bacteria were grown overnight in NYG medium, adjusted to OD_600nm = 0.6, and then subjected to the indicated treatment for 24 h before final measurements. Bacterial populations (CFU/ml) of bacterial culture in the presence of sinapaldehyde (Sigma-Aldrich) or eugenol (Sigma-Aldrich) were measured. Eugenol was used as a positive antibiotic control.

Statistical analysis

Statistical analysis was performed using either Student's t test or Welch's t test, as indicated in the figure legends. The sample sizes (n) used for this study and relevant P values are also given in the figures.

Acknowledgments

The authors are grateful to Bart lab members for helpful discussions.