Arabidopsis non-host resistance PSS30 gene enhances broad-spectrum disease resistance in the soybean cultivar Williams 82

Non-host resistance (NHR), which protects all members of a plant species from non-adapted or non-host plant pathogens, is the most common form of plant immunity. NHR provides the most durable and robust form of broad-spectrum immunity against non-adaptive pathogens pathogenic to other crop species. In a mutant screen for loss of Arabidopsis (Arabidopsis thaliana) NHR against the soybean (Glycine max (L.) Merr.) pathogen Phytophthora sojae, the Phytophthora sojae-susceptible 30 (pss30) mutant was identified. The pss30 mutant is also susceptible to the soybean pathogen Fusarium virguliforme. PSS30 encodes a folate transporter, AtFOLT1, which was previously localized to chloroplasts and implicated in the transport of folate from the cytosol to plastids. We show that two Arabidopsis folate biosynthesis mutants with reduced folate levels exhibit a loss of non-host immunity against P. sojae. As compared to the wild-type Col-0 ecotype, the steady-state folate levels are reduced in the pss1, atfolt1 and two folate biosynthesis mutants, suggesting that folate is required for non-host immunity. Overexpression of AtFOLT1 enhances immunity of transgenic soybean lines against two serious soybean pathogens, the fungal pathogen F. virguliforme and the soybean cyst nematode (SCN) Heterodera glycines. Transgenic lines showing enhanced SCN resistance also showed increased levels of folate accumulation. This study thus suggests that folate contributes to non-host plant immunity and that overexpression of a non-host resistance gene could be a suitable strategy for generating broad-spectrum disease resistance in crop plants.     

Characterization of nonhost resistance of Arabidopsis to the Asian soybean rust

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is a devastating disease of soybean. We report the use of the nonhost plant Arabidopsis thaliana to identify the genetic basis of resistance to P. pachyrhizi. Upon attack by P. pachyrhizi, epidermal cells of wild-type Arabidopsis accumulated H2O2, which likely orchestrates the frequently observed epidermal cell death. However, even when epidermal cell death occurred, fungal hyphae grew on and infection was terminated at the mesophyll boundary. These events were associated with expression of PDF1.2, suggesting that P. pachyrhizi, an ostensible biotroph, mimics aspects of a necrotroph. Extensive colonization of the mesophyll occurred in Arabidopsis pen mutants with defective penetration resistance. Although haustoria were found occasionally in mesophyll cells, the successful establishment of biotrophy failed, as evidenced by the cessation of fungal growth. Double mutants affected in either jasmonic acid or salicylic acid signaling in the pen3-1 background revealed the involvement of both pathways in nonhost resistance (NHR) of Arabidopsis to P. pachyrhizi. Interestingly, expression of AtNHL10, a gene that is expressed in tissue undergoing the hypersensitive response, was only triggered in infected pen3-1 mutants. Thus, a suppression of P. pachyrhizi-derived effectors by PEN3 can be inferred. Our results demonstrate that Arabidopsis can be used to study mechanisms of NHR to ASR.     

The PEN1 syntaxin defines a novel cellular compartment upon fungal attack and is required for the timely assembly of papillae

Attack by the host powdery mildew Erysiphe cichoracearum usually results in successful penetration and rapid proliferation of the fungus on Arabidopsis. By contrast, the nonhost barley powdery mildew Blumeria graminis f. sp. hordei (Bgh) typically fails to penetrate Arabidopsis epidermal cells. In both instances the plant secretes cell wall appositions or papillae beneath the penetration peg of the fungus. Genetic screens for mutations that result in increased penetration of Bgh on Arabidopsis have recently identified the PEN1 syntaxin. Here we examine the role of PEN1 and of its closest homologue, SYP122, identified as a syntaxin whose expression is responsive to infection. pen1 syp122 double mutants are both dwarfed and necrotic, suggesting that the two syntaxins have overlapping functions. Although syp122-1 and the cell wall mur mutants have considerably more pronounced primary cell wall defects than pen1 mutants, these have relatively subtle or no effects on penetration resistance. Upon fungal attack, PEN1 appears to be actively recruited to papillae, and there is a 2-h delay in papillae formation in the pen1-1 mutant. We conclude that SYP122 may have a general function in secretion, including a role in cell wall deposition. By contrast, PEN1 appears to have a basal function in secretion and a specialized defense-related function, being required for the polarized secretion events that give rise to papilla formation.     

拟南芥与大豆疫霉菌的非寄主互作及一个感病突变体的遗传分析

非寄主抗病性是一种普遍的自然现象,该文通过建立拟南芥．大豆疫霉菌（Arabidopsis thaliana—Phytophthora sojae）非寄主互作系统,筛选对大豆疫霉菌感病的拟南芥突变体,为研究植物对卵菌的非寄主抗病性遗传机制奠定基础。以大豆疫霉菌游动孢子接种拟南芥T—DNA插入突变体离体叶片,从代表12000个独立转化株系的40000株T3代T。DNA插入拟南芥突变体中获得一系列对大豆疫霉菌感病的突变体。其中突变体581-51感病性状表现稳定,离体叶片接菌后3天内出现明显的水渍状病斑,4—5天后产生大量卵孢子和／或孢子囊。细胞学观察发现有典型的吸器形成。Southern杂交和遗传分析结果表明,581—51突变体含有4个T-DNA插入事件,其感病性状可能由隐性单基因控制。     

Interspecies gene transfer provides soybean resistance to a fungal pathogen

Fungal pathogens pose a major challenge to global crop production. Crop varieties that resist disease present the best defence and offer an alternative to chemical fungicides. Exploiting durable nonhost resistance (NHR) for crop protection often requires identification and transfer of NHR‐linked genes to the target crop. Here, we identify genes associated with NHR of Arabidopsis thaliana to Phakopsora pachyrhizi, the causative agent of the devastating fungal disease called Asian soybean rust. We transfer selected Arabidopsis NHR‐linked genes to the soybean host and discover enhanced resistance to rust disease in some transgenic soybean lines in the greenhouse. Interspecies NHR gene transfer thus presents a promising strategy for genetically engineered control of crop diseases.     

Arabidopsis PEN3/PDR8, an ATP binding cassette transporter, contributes to nonhost resistance to inappropriate pathogens that enter by direct penetration

Arabidopsis thaliana is a host to the powdery mildew Erysiphe cichoracearum and nonhost to Blumeria graminis f. sp hordei, the powdery mildew pathogenic on barley (Hordeum vulgare). Screening for Arabidopsis mutants deficient in resistance to barley powdery mildew identified PENETRATION3 (PEN3). pen3 plants permitted both increased invasion into epidermal cells and initiation of hyphae by B. g. hordei, suggesting that PEN3 contributes to defenses at the cell wall and intracellularly. pen3 mutants were compromised in resistance to the necrotroph Plectosphaerella cucumerina and to two additional inappropriate biotrophs, pea powdery mildew (Erysiphe pisi) and potato late blight (Phytophthora infestans). Unexpectedly, pen3 mutants were resistant to E. cichoracearum. This resistance was salicylic acid-dependent and correlated with chlorotic patches. Consistent with this observation, salicylic acid pathway genes were hyperinduced in pen3 relative to the wild type. The phenotypes conferred by pen3 result from the loss of function of PLEIOTROPIC DRUG RESISTANCE8 (PDR8), a highly expressed putative ATP binding cassette transporter. PEN3/PDR8 tagged with green fluorescent protein localized to the plasma membrane in uninfected cells. In infected leaves, the protein concentrated at infection sites. PEN3/PDR8 may be involved in exporting toxic materials to attempted invasion sites, and intracellular accumulation of these toxins in pen3 may secondarily activate the salicylic acid pathway.     

拟南芥与大豆疫霉菌的非寄主互作及一个感病突变体的遗传分析

非寄主抗病性是一种普遍的自然现象, 该文通过建立拟南芥-大豆疫霉菌(Arabidopsis thaliana-Phytophthora sojae)非寄主互作系统, 筛选对大豆疫霉菌感病的拟南芥突变体, 为研究植物对卵菌的非寄主抗病性遗传机制奠定基础。以大豆疫霉菌游动孢子接种拟南芥T-DNA插入突变体离体叶片, 从代表12 000个独立转化株系的40 000株T₃代T-DNA插入拟南芥突变体中获得一系列对大豆疫霉菌感病的突变体。其中突变体581-51感病性状表现稳定, 离体叶片接菌后3天内出现明显的水渍状病斑, 4–5天后产生大量卵孢子和/或孢子囊。细胞学观察发现有典型的吸器形成。Southern杂交和遗传分析结果表明, 581-51突变体含有4个T-DNA插入事件, 其感病性状可能由隐性单基因控制。     

Plant resistance signalling hijacked by a necrotrophic fungal pathogen

The strategies used by necrotrophic fungal pathogens to infect plants are often perceived as lacking the sophistication of their haustorium producing, host defence suppressing, biotrophic counterparts. There is also a relative paucity of knowledge regarding how effective gene-for-gene based resistance reactions might function against necrotrophic plant pathogens. However, recent data has emerged from a number of systems which has highlighted that particular species of necrotrophic (and/or hemibiotrophic) fungi, have evolved very sophisticated strategies for plant infection which appear, in fact, to hijack the host resistance responses that are commonly deployed against biotrophs. Both disease resistance (R) protein homologues and mitogen-activated protein kinase (MAPK) cascades commonly associated with incompatible disease resistance responses; appear to be targeted by necrotrophic fungi during compatible disease interactions. These findings highlight an emerging sophistication in the strategies deployed by necrotrophic fungi to infect plants.Key words: Mycosphaerella graminicola, Septoria tritici, Triticum aestivum, mitogen-activated protein kinase, programmed cell death, fungal pathogen, disease resistance, disease susceptibility, toxin     

Phytosterols play a key role in plant innate immunity against bacterial pathogens by regulating nutrient efflux into the apoplast

Bacterial pathogens colonize a host plant by growing between the cells by utilizing the nutrients present in apoplastic space. While successful pathogens manipulate the plant cell membrane to retrieve more nutrients from the cell, the counteracting plant defense mechanism against nonhost pathogens to restrict the nutrient efflux into the apoplast is not clear. To identify the genes involved in nonhost resistance against bacterial pathogens, we developed a virus-induced gene-silencing-based fast-forward genetics screen in Nicotiana benthamiana. Silencing of N. benthamiana SQUALENE SYNTHASE, a key gene in phytosterol biosynthesis, not only compromised nonhost resistance to few pathovars of Pseudomonas syringae and Xanthomonas campestris, but also enhanced the growth of the host pathogen P. syringae pv tabaci by increasing nutrient efflux into the apoplast. An Arabidopsis (Arabidopsis thaliana) sterol methyltransferase mutant (sterol methyltransferase2) involved in sterol biosynthesis also compromised plant innate immunity against bacterial pathogens. The Arabidopsis cytochrome P450 CYP710A1, which encodes C22-sterol desaturase that converts β-sitosterol to stigmasterol, was dramatically induced upon inoculation with nonhost pathogens. An Arabidopsis Atcyp710A1 null mutant compromised both nonhost and basal resistance while overexpressors of AtCYP710A1 enhanced resistance to host pathogens. Our data implicate the involvement of sterols in plant innate immunity against bacterial infections by regulating nutrient efflux into the apoplast.     

Glutathione and tryptophan metabolites are key players in Arabidopsis nonhost resistance against Colletotrichum gloeosporioides

The nonhost resistance of Arabidopsis against hemibiotrophic fungi in the genus Colletotrichum consists of pre- and post-invasive immune responses. Previously, we reported EDR1 and PEN2 as important components of Arabidopsis pre-invasive resistance toward non-adapted Colletotrichum gloeosporioides (Cg). However, despite their defect in entry control pen2 and edr1 mutants terminated further growth of this pathogen by activating the post-invasive hypersensitive response (HR) accompanied by plant cell death. In the present study, we showed that γ-glutamylcysteine synthetase (GSH1), which is required for glutathione biosynthesis, and tryptophan (Trp) metabolism contribute to pre- and post-invasive non-host resistance against Cg. We found GSH1 to be involved in the PEN2-dependent entry control of Cg. Opposite to pen2 and edr1, gsh1 mutants failed to restrict the invasive growth of the pathogen, which demonstrated the requirement for GSH1 during post-invasive non-host resistance. Based on the infection and metabolic phenotypes of Arabidopsis mutants defective in Trp metabolism, we showed that the biosynthesis of Trp-derived phytochemicals is also essential for resistance to Cg during the post-invasive HR. By contrast, GSH1 and these metabolites are dispensable for the induction of HR cell death, which is triggered in the non-invaded mesophyll cells adjacent to the Cg-invaded epidermal cells.     

RCH1, a locus in Arabidopsis that confers resistance to the hemibiotrophic fungal pathogen Colletotrichum higginsianum

When challenged with the crucifer pathogen Colletotrichum higginsianum, Arabidopsis thaliana ecotype Columbia (Col-0) was colonized by the fungus within 2 to 3 days, developing brown necrotic lesions surrounded by a yellow halo. Lesions spread from the inoculation site within 3 to 4 days, and subsequently continued to expand until they covered the entire leaf. Electron microscopy confirmed that C. higginsianum is a hemibiotroph on Arabidopsis, feeding initially on living cells as a biotroph before switching to a necrotrophic mode of growth. A collection of 37 ecotypes of Arabidopsis varied in their responses to infection by C. higginsianum. The ecotype Eil-0 was highly resistant, with symptoms limited to necrotic flecking and with only very limited fungal colonization. Analyses suggested that the hypersensitive response and reactive oxygen species may be important in this defense response. Expression analyses with cDNA microarrays indicated that the defense reaction depends primarily on the jasmonic acid- and ethylene-dependent signaling pathways and, to a lesser extent, on the salicylate-dependent pathway. Crosses between the Eil-0 and Col-0 ecotypes suggested that the resistance in Eil-0 was dominant and was conferred by a single locus, which we named RCH1. RCH1 is the first resistance locus to be identified from Arabidopsis against the hemibiotrophic fungus genus Colletotrichum.     

Nonhost resistance in Arabidopsis-Colletotrichum interactions acts at the cell periphery and requires actin filament function

Pathogenesis of nonadapted fungal pathogens is often terminated coincident with their attempted penetration into epidermal cells of nonhost plants. The genus Colletotrichum represents an economically important group of fungal plant pathogens that are amenable to molecular genetic analysis. Here, we investigated interactions between Arabidopsis and Colletotrichum to gain insights in plant and pathogen processes activating nonhost resistance responses. Three tested nonadapted Colletotrichum species differentiated melanized appressoria on Arabidopsis leaves but failed to form intracellular hyphae. Plant cells responded to Colletotrichum invasion attempts by the formation of PMR4/GSL5-dependent papillary callose. Appressorium differentiation and melanization were insufficient to trigger this localized plant cell response, but analysis of nonpathogenic C. lagenarium mutants implicates penetration-peg formation as the inductive cue. We show that Arabidopsis PEN1 syntaxin controls timely accumulation of papillary callose but is functionally dispensable for effective preinvasion (penetration) resistance in nonhost interactions. Consistent with this observation, green fluorescent protein-tagged PEN1 did not accumulate at sites of attempted penetration by either adapted or nonadapted Colletotrichum species, in contrast to the pronounced focal accumulations of PEN1 associated with entry of powdery mildews. We observed extensive reorganization of actin microfilaments leading to polar orientation of large actin bundles towards appressorial contact sites in interactions with the nonadapted Colletotrichum species. Pharmacological inhibition of actin filament function indicates a functional contribution of the actin cytoskeleton for both preinvasion resistance and papillary callose formation. Interestingly, the incidence of papilla formation at entry sites was greatly reduced in interactions with C. higginsianum isolates, indicating that this adapted pathogen may suppress preinvasion resistance at the cell periphery.     

A rice gene encoding glycosyl hydrolase plays contrasting roles in immunity depending on the type of pathogens

Because pathogens use diverse infection strategies, plants cannot use one-size-fits-all defence and modulate defence responses based on the nature of pathogens and pathogenicity mechanism. Here, we report that a rice glycoside hydrolase (GH) plays contrasting roles in defence depending on whether a pathogen is hemibiotrophic or necrotrophic. The Arabidopsis thaliana MORE1 (M agnaporthe o ryzae re sistance 1) gene, encoding a member of the GH10 family, is needed for resistance against M. oryzae and Alternaria brassicicola, a fungal pathogen infecting A. thaliana as a necrotroph. Among 13 rice genes homologous to MORE1, 11 genes were induced during the biotrophic or necrotrophic stage of infection by M. oryzae. CRISPR/Cas9-assisted disruption of one of them (OsMORE1a) enhanced resistance against hemibiotrophic pathogens M. oryzae and Xanthomonas oryzae pv. oryzae but increased susceptibility to Cochliobolus miyabeanus, a necrotrophic fungus, suggesting that OsMORE1a acts as a double-edged sword depending on the mode of infection (hemibiotrophic vs. necrotrophic). We characterized molecular and cellular changes caused by the loss of MORE1 and OsMORE1a to understand how these genes participate in modulating defence responses. Although the underlying mechanism of action remains unknown, both genes appear to affect the expression of many defence-related genes. Expression patterns of the GH10 family genes in A. thaliana and rice suggest that other members also participate in pathogen defence.     

Haemoglobin modulates salicylate and jasmonate/ethylene-mediated resistance mechanisms against pathogens

Nitric oxide (NO) plays a role in defence against hemibiotrophic pathogens mediated by salicylate (SA) and also necrotrophic pathogens influenced by jasmonate/ethylene (JA/Et). This study examined how NO-oxidizing haemoglobins (Hb) encoded by GLB1, GLB2, and GLB3 in Arabidopsis could influence both defence pathways. The impact of Hb on responses to the hemibiotrophic Pseudomonas syringae pathovar tomato (Pst) AvrRpm1 and the necrotrophic Botrytis cinerea were investigated using glb1, glb2, and glb3 mutant lines and also CaMV 35S GLB1 and GLB2 overexpression lines. In glb1, but not glb2 and glb3, increased resistance was observed to both pathogens but was compromised in the 35S-GLB1. A quantum cascade laser-based sensor measured elevated NO production in glb1 infected with Pst AvrRpm1 and B. cinerea, which was reduced in 35S-GLB1 compared to Col-0. SA accumulation was increased in glb1 and reduced in 35S-GLB1 compared to controls following attack by Pst AvrRpm1. Similarly, JA and Et levels were increased in glb1 but decreased in 35S-GLB1 in response to attack by B. cinerea. Quantitative PCR assays indicated reduced GLB1 expression during challenge with either pathogen, thus this may elevate NO concentration and promote a wide-ranging defence against pathogens.     

SOBIR1 and AGB1 independently contribute to nonhost resistance to Pyricularia oryzae (syn. Magnaporthe oryzae) in Arabidopsis thaliana

ABSTRACT

Rice blast caused by Pyricularia oryzae (syn. Magnaporthe oryzae) is a disease devastating to rice. We have studied the Arabidopsis-P. oryzae pathosystem as a model system for nonhost resistance (NHR) and found that SOBIR1, but not BAK1, is a positive regulator of NHR to P. oryzae in Arabidopsis. AGB1 is also involved in NHR. However, the genetic interactions between SOBIR1, BAK1, and AGB1 are uncharacterized. In this study, we delineated the genetic interactions between SOBIR1, BAK1, and AGB1 in NHR to P. oryzae in Arabidopsis and found SOBIR1 and AGB1 independently control NHR to P. oryzae in Arabidopsis pen2-1 mutant plants. Furthermore, XLG2, but not TMM, has a positive role in penetration resistance to P. oryzae in Arabidopsis pen2-1 mutant plants. Our study characterized genetic interactions in Arabidopsis NHR.

Abbreviations: PRR: pattern recognition receptor, RLK: receptor-like kinase, RLP: receptor-like protein, BAK1: BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1, BIR1: BAK1-INTERACTING RECEPTOR-LIKE KINASE 1, SOBIR1: SUPPRESSOR OF BIR1-1-1, AGB1: ARABIDOPSIS G PROTEIN ß-SUBUNIT 1, XLG2: EXTRA-LARGE G PROTEIN 2     

Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean.

To develop a model system for molecular genetic analysis of plant-pathogen interactions, we studied the interaction between Arabidopsis thaliana and the bacterial pathogen Pseudomonas syringae pv tomato (Pst). Pst strains were found to be virulent or avirulent on specific Arabidopsis ecotypes, and single ecotypes were resistant to some Pst strains and susceptible to others. In many plant-pathogen interactions, disease resistance is controlled by the simultaneous presence of single plant resistance genes and single pathogen avirulence genes. Therefore, we tested whether avirulence genes in Pst controlled induction of resistance in Arabidopsis. Cosmids that determine avirulence were isolated from Pst genomic libraries, and the Pst avirulence locus avrRpt2 was defined. This allowed us to construct pathogens that differed only by the presence or absence of a single putative avirulence gene. We found that Arabidopsis ecotype Col-0 was susceptible to Pst strain DC3000 but resistant to the same strain carrying avrRpt2, suggesting that a single locus in Col-0 determines resistance. As a first step toward genetically mapping the postulated resistance locus, an ecotype susceptible to infection by DC3000 carrying avrRpt2 was identified. The avrRpt2 locus from Pst was also moved into virulent strains of the soybean pathogen P. syringae pv glycinea to test whether this locus could determine avirulence on soybean. The resulting strains induced a resistant response in a cultivar-specific manner, suggesting that similar resistance mechanisms may function in Arabidopsis and soybean.     

Activity determinants and functional specialization of Arabidopsis PEN1 syntaxin in innate immunity

In eukaryotes, proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family are believed to have a general role for the fusion of intracellular transport vesicles with acceptor membranes. Arabidopsis thaliana PEN1 syntaxin resides in the plasma membrane and was previously shown to act together with its partner SNAREs, the adaptor protein SNAP33, and endomembrane-anchored VAMP721/722 in the execution of secretory immune responses against powdery mildew fungi. We conducted a structure-function analysis of PEN1 and show that N-terminal phospho-mimicking and non-phosphorylatable variants neither affected binary nor ternary SNARE complex formation with cognate partners in vitro. However, expression of these syntaxin variants at native protein levels in a pen1 mutant background suggests that phosphorylation is required for full resistance activity in planta. All tested site-directed substitutions of SNARE domain or "linker region" residues reduced PEN1 defense activity. Two of the variants failed to form ternary complexes with the partner SNAREs in vitro, possibly explaining their diminished in planta activity. However, impaired pathogen defense in plants expressing a linker region variant is likely because of PEN1 destabilization. Although Arabidopsis PEN1 and SYP122 syntaxins share overlapping functions in plant growth and development, PEN1 activity in disease resistance is apparently the result of a complete functional specialization. Our findings are consistent with the hypothesis that PEN1 acts in plant defense through the formation of ternary SNARE complexes and point to the existence of unknown regulatory factors. Our data indirectly support structural inferences that the four-helical coiled coil bundle in ternary SNARE complexes is formed in a sequential order from the N- to C-terminal direction.     

PHOSPHATIDYLSERINE SYNTHASE1 is required for microspore development in Arabidopsis thaliana

Phosphatidylserine (PS) has many important biological roles, but little is known about its role in plants, partly because of its low abundance. We show here that PS is enriched in Arabidopsis floral tissues and that genetic disruption of PS biosynthesis decreased heterozygote fertility due to inhibition of pollen maturation. At1g15110, designated PSS1, encodes a base-exchange-type PS synthase. Escherichia coli cells expressing PSS1 accumulated PS in the presence of l-serine at 23°C. Promoter-GUS assays showed PSS1 expression in developing anther pollen and tapetum. A few seeds with pss1-1 and pss1-2 knockout alleles escaped embryonic lethality but developed into sterile dwarf mutant plants. These plants contained no PS, verifying that PSS1 is essential for PS biosynthesis. Reciprocal crossing revealed reduced pss1 transmission via male gametophytes, predicting a rate of 61.6%pss1-1 pollen defects in PSS1/pss1-1 plants. Alexander's staining of inseparable qrt1-1 PSS1/pss1-1 quartets revealed a rate of 42% having three or four dead pollen grains, suggesting sporophytic pss1-1 cell death effects. Analysis with the nuclear stain 4',6-diamidino-2-phenylindole (DAPI) showed that all tetrads from PSS1/pss1-1 anthers retain their nuclei, whereas unicellular microspores were sometimes anucleate. Transgenic Arabidopsis expressing a GFP-LactC2 construct that binds PS revealed vesicular staining in tetrads and bicellular microspores and nuclear membrane staining in unicellular microspores. Hence, distribution and/or transport of PS across membranes were dynamically regulated in pollen microspores. However, among unicellular microspores from PSS1/pss1-2 GFP-LactC2 plants, all anucleate microspores showed little GFP-LactC2 fluorescence, suggesting that pss1-2 microspores are more sensitive to sporophytic defects or show partial gametophytic defects.