Hpa2 Required by HrpF To Translocate Xanthomonas oryzae Transcriptional Activator-Like Effectors into Rice for Pathogenicity

Abstract

Xanthomonas oryzae pv. oryzicola, the causative agent of bacterial leaf streak, injects a plethora of effectors through the type III secretion system (T3SS) into rice cells to cause disease. The T3SS, encoded by the hrp genes, is essential for the pathogen to elicit the hypersensitive response (HR) in nonhost tobacco and for pathogenicity in host rice. Whether or not a putative lytic transglycosylase, Hpa2, interacts with a translocon protein, HrpF, to facilitate bacterial pathogenicity remains unknown. Here we demonstrated that both the hpa2 and hrpF genes are required for the pathogenicity of X. oryzae pv. oryzicola strain RS105 in rice but not for HR induction in tobacco. The expression of hpa2 was positively regulated by HrpG and HrpD6 but not by HrpX. In vivo secretion and subcellular localization analyses confirmed that Hpa2 secretion is dependent on HpaB (a T3SS exit protein) and that Hpa2 binds to the host cell membrane. Protein-protein assays demonstrated that Hpa2 interacts with HrpF. In planta translocation of AvrXa10 indicated that the mutation in hpa2 and hrpF inhibits the injection of the HpaB-dependent transcriptional activator-like (TAL) effector into rice. These findings suggest that Hpa2 and HrpF form a complex to translocate T3S effectors into plant cells for pathogenesis in host rice.Xanthomonas oryzae pv. oryzicola, the causative agent of bacterial leaf streak disease in rice, is one of the model organisms for studying the molecular mechanisms of plant-pathogen pathosystems (41, 59). The bacterial ability to trigger the hypersensitive response (HR), a rapid and localized programmed cell death in nonhosts or in resistant hosts, and to be pathogenic in host plants depends on a type III secretion system (T3SS) encoded by a 27-kb hrp cluster containing 10 hrp, 9 hrc (hrp-conserved), and 8 hpa (hrp-associated) genes according to the homologous regions in other Xanthomonas species (9,41,59). Some of the hrp-hrc-hpa gene products comprise a pedestal-like T3SS structure that traverses the two bacterial membranes (21, 24), a pilus-like secretion channel (HrpE) outside HrcC (52), and a translocon protein (HrpF) in the eukaryotic host membrane (3, 4, 6, 21, 48). As a whole, the T3SS apparatus injects a number of effectors into the apoplast and cytosol of eukaryotic host cells, including harpins, which elicit HR induction in nonhost apoplasts (4, 45, 58), and transcriptional activator-like (TAL) effectors, which lead to disease susceptibility in hosts or trigger disease resistance in nonhosts upon interaction with a specific R gene product surveillance system (4,11,38,39,40,49,56). The virulence of Xanthomonas oryzae pv. oryzae is markedly reduced when hrpF is mutated (48). However, hrpF mutation has not been investigated in X. oryzae pv. oryzicola.The hpa genes contribute to virulence, but strains with mutations in hpa genes generally do not exhibit phenotypic changes in disease symptoms of the same severity as those with other hrp-hrc gene mutations (9,25,29). Some Hpa proteins, such as HpaB and HpaC ...

Protein Export According to Schedule: Architecture, Assembly, and Regulation of Type III Secretion Systems from Plant- and Animal-Pathogenic Bacteria

“…Interestingly, Hpa2 from X. oryzae pv. oryzicola contributes to effector protein secretion and interacts with the translocon protein HrpF, suggesting that it not only acts as an LT but also plays a role at the host-pathogen interface (319). The predicted LTs HrpH and HopP1 from P. syringae are even translocated by the T3S system into the plant cell and were shown to suppress basal plant defense responses in Nicotiana benthamiana (412,413).…”

“…Some predicted LTs that are involved in T3S, including Hpa2 from the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzicola as well as HrpH and HopP1 from P. syringae, are themselves secreted, possibly to prevent further LT-mediated peptidoglycan degradation after the assembly of the secretion apparatus (319,412) (Table 3). Interestingly, Hpa2 from X. oryzae pv.…”

The Predicted Lytic Transglycosylase HpaH from Xanthomonas campestris pv. vesicatoria Associates with the Type III Secretion System and Promotes Effector Protein Translocation

“…The periplasm is presumably not the final destination of HpaH, suggesting that its association with peptidoglycan and components of the T3S system is only transient. As was reported for other LTs, HpaH is released into the extracellular milieu, possibly to avoid a periplasmic accumulation of HpaH and thus deleterious effects resulting from enhanced local degradation of peptidoglycan (17,18). Secreted putative LTs include HrpH and HopP1 from P. syringae, which are translocated into the plant cell (17).…”

“…To date, a virulence function has been described for putative LTs from enterohemorrhagic Escherichia coli (EHEC), enteropathogenic Escherichia coli (EPEC), and plant-pathogenic bacteria, including pathovars of Pseudomonas syringae and Xanthomonas spp. (14)(15)(16)(17)(18)(19)(20)(21). In most cases, however, the enzymatic activity of these proteins and their contribution to the assembly of the T3S system have not yet been experimentally confirmed.…”

KdgR, an IClR Family Transcriptional Regulator, Inhibits Virulence Mainly by Repression of hrp Genes in Xanthomonas oryzae pv. oryzae

“…So far, many genes of X. oryzae pv. oryzae have been suggested to be associated with pathogenesis (9,10,20), and many regulatory proteins, such as OryR, PecS, and LrpX (18,26,35), have been shown to be involved in regulation of these pathogenicity-related genes.…”